Screening method

ABSTRACT

The present invention provides a method of screening a mammal for the onset or predisposition to the onset of a neuropsychiatric disorder. More particularly, the present invention provides a method of screening a mammal for the onset or predisposition to the onset of schizophrenia by screening for a decrease in the functional level of protein 1 4-3-3ζ. In a related aspect, the present invention also provides a means of monitoring a patient diagnosed with a neuropsychiatric disorder, such as schizophrenia, by screening for changes to functional levels of protein 14-3-3 ζ. This may be useful, for example, in the context of evaluating the effectiveness of a prophylactic or therapeutic treatment regime or otherwise monitoring the impact of physiological or metabolic changes which may occur in a patient. The method of the present invention is useful in a wide range of applications including, inter alia, providing a means of identifying mammals susceptible to the onset of a neuropsychiatric condition, such as a condition characterized by one or more symptoms of schizophrenia, thereby enabling the implementation of prophylactic or early therapeutic intervention in an effort to either minimize or prevent the onset of the condition. It also provides a means of confirming diagnoses which would otherwise be based solely on an assessment of positive and negative symptoms.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 14/346,292, filed Mar. 20, 2014, which is a U.S. National PhaseApplication of PCT International Application Number PCT/AU2012/001141,filed on Sep. 21, 2012, designating the United States of America andpublished in the English language, which is an International Applicationof and claims the benefit of priority to Australian Patent ApplicationNo. 2011903906, filed Sep. 22, 2011. The disclosures of theabove-referenced applications are hereby expressly incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention provides a method of screening a mammal for theonset or predisposition to the onset of a neuropsychiatric disorder.More particularly, the present invention provides a method of screeninga mammal for the onset or predisposition to the onset of schizophreniaby screening for a decrease in the functional level of protein 14-3-3ζ.In a related aspect, the present invention also provides a means ofmonitoring a patient diagnosed with a neuropsychiatric disorder, such asschizophrenia, by screening for changes to functional levels of protein14-3-3ζ. This may be useful, for example, in the context of evaluatingthe effectiveness of a prophylactic or therapeutic treatment regime orotherwise monitoring the impact of physiological or metabolic changeswhich may occur in a patient. The method of the present invention isuseful in a wide range of applications including, inter alia, providinga means of identifying mammals susceptible to the onset of aneuropsychiatric condition, such as a condition characterised by one ormore symptoms of schizophrenia, thereby enabling the implementation ofprophylactic or early therapeutic intervention in an effort to eitherminimise or prevent the onset of the condition. It also provides a meansof confirming diagnoses which would otherwise be based solely on anassessment of positive and negative symptoms.

In a related aspect, the present invention also provides an animal modelwhich is useful, inter alia, for screening for or evaluating agents foruse in treating a neuropsychiatric condition.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Schizophrenia is one of the most disabling and emotionally devastatingillnesses known to man. Unfortunately, because it has been misunderstoodfor so long, it has received relatively little attention and its victimshave been undeservingly stigmatized. Schizophrenia is, in fact, a fairlycommon disorder. It affects both sexes equally and strikes about 1% ofthe population worldwide. Another 2-3% have schizotypal personalitydisorder, a milder form of the disease. Because of its prevalence andseverity, schizophrenia has been studied extensively in an effort todevelop better criteria for diagnosing the illness.

Schizophrenia is characterized by a constellation of distinctive andpredictable symptoms. The symptoms that are most commonly associatedwith the disease are called positive symptoms, that denote the presenceof grossly abnormal behaviour. These include thought disorder (speechwhich is difficult to follow or jumping from one subject to another withno logical connection), delusions (false beliefs of persecution, guilt,grandeur or being under outside control) and hallucinations (visual orauditory). Thought disorder is the diminished ability to think clearlyand logically. Often it is manifested by disconnected and nonsensicallanguage that renders the person with schizophrenia incapable ofparticipating in conversation, contributing to his alienation from hisfamily, friends and society. Delusions are common among individuals withschizophrenia. An affected person may believe that he is being conspiredagainst (called “paranoid delusion”). “Broadcasting” describes a type ofdelusion in which the individual with this illness believes that histhoughts can be heard by others. Hallucinations can be heard, seen oreven felt. Most often they take the form of voices heard only by theafflicted person. Such voices may describe the person's actions, warnhim of danger or tell him what to do. At times the individual may hearseveral voices carrying on a conversation. Less obvious than the above“positive symptoms” and “thought disorder” but equally serious are thedeficit or negative symptoms that represent the absence of normalbehaviour. These include flat or blunted affect (i.e. lack of emotionalexpression), apathy, social withdrawal and lack of insight.

The onset of schizophrenia usually occurs during adolescence or earlyadulthood, although it has been known to develop in older people. Onsetmay be rapid with acute symptoms developing over several weeks, or itmay be slow developing over months or even years. While schizophreniacan affect anyone at any point in life, it is somewhat more common inthose persons who are genetically predisposed to the disease with thefirst psychotic episode generally occurring in late adolescence or earlyadulthood. The probability of developing schizophrenia as the offspringof two parents, neither of whom has the disease is 1 percent. Theprobability of developing schizophrenia as the offspring of one parentwith the disease is approximately 13 percent. The probability ofdeveloping schizophrenia as the offspring of both parents with thedisease is approximately 35 percent. This is indicative of the existenceof a genetic link.

Three-quarters of persons with schizophrenia develop the disease between16 and 25 years of age. Onset is uncommon after age 30 and rare afterage 40. In the 16-25 year old age group, schizophrenia affects more menthan women. In the 25-30 year old group, the incident is higher in womenthan in men.

In general, the study of any illness requires that there should be goodcriteria for diagnosis. In fact, diagnosis should ultimately be based oncauses i.e., on whether an illness results from a genetic defect, aviral or bacterial infection, toxins or stress. Unfortunately, thecauses of most psychiatric illnesses are unknown and therefore thesedisorders are still grouped according to which of the four major mentalfaculties are affected:

(i) disorders of thinking and cognition

(ii) disorders of mood

(iii) disorders of social behaviour; and

(iv) disorders of learning, memory and intelligence.

Accordingly, since so little is known of the biological causes of theseconditions, there is an ongoing need to elucidate the mechanisms bywhich these diseases are induced and progress.

The 14-3-3 proteins constitute a family of highly conserved regulatorymolecules expressed abundantly throughout development and in adulttissue. These proteins comprise seven distinct isoforms (β, ζ, ε, γ, η,τ, σ), that bind a multitude of functionally diverse signallingmolecules to control cell cycle regulation, proliferation, migration,differentiation and apoptosis (Berg et al. Nat Rev Neurosci 2003:4(9):752-762; Fu et al. Annu Rev Pharmacol Toxical 2000; 40:617-647;Toyo-oka et al. Nat Genet 2003 July; 34(3): 274-285: Aitken A. SeminCancer Biol 2006; 16(3):162-172: Rosner et al. Amino Acids 2006;30(1):105-109).

To date, the role, if any, of the protein 14-3-3 family of molecules inschizophrenia has remained elusive. Some research has focused, albeit sofar inconclusively, on identifying single nuclear polymorphismsassociated with a predisposition to developing a neuropsychiatriccondition such as schizophrenia. Studies aimed at investigating changesto levels of protein 14-3-3 isoforms, irrespective of whether or notthose molecules are mutated, have tended to focus on changes to thelevels of the eta and theta isoforms, although to date there has notbeen any conclusive-evidence that they are reliable markers of the onsetof a neuropsychiatric condition. In relation to other of the protein14-3-3 isoforms, such as beta and zeta, Wong et al. (2005) found nochange to expression levels in schizophrenia and bipolar disorders.Middleton et al. (2005) went further and stated that these particularisoforms are not likely to be directly related to a genetic risk fordeveloping schizophrenia and that neither marker provides a strongassociation with schizophrenia.

Nevertheless, and contrary to these findings, in work leading up to thepresent invention it has been determined that a reduction in thefunctional level of protein 14-3-3ζ is associated with the onset of orpredisposition to the onset of a neuropsychiatric disorder, such as acondition which is characterised by one or more symptoms ofschizophrenia. Still further, it has also been determined that areduction in the level of protein 14-3-3ζ/DISC1 complex formation issimilarly diagnostic. These findings have now facilitated the design ofmethodology to routinely and accurately screen individuals to confirmthe onset of, or a predisposition to the development of, aneuropsychiatric disorder.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein, the term “derived from” shall be taken to indicate thata particular integer or group of integers has originated from thespecies specified, but has not necessarily been obtained directly fromthe specified source. Further, as used herein the singular forms of “a”,“and” and “the” include plural referents unless the context clearlydictates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Those skilled in the art will be aware that the invention describedherein is subject to variations and modifications other than thosespecifically described. It is to be understood that the inventiondescribed herein includes all such variations and modifications. Theinvention also includes all such steps, features, compositions andcompounds referred to or indicated in this specification, individuallyor collectively, and any and all combinations of any two or more of saidsteps or features.

The subject specification contains nucleotide sequence informationprepared using the programme PatentIn Version 3.5, presented hereinafter the bibliography. Each nucleotide sequence is identified in thesequence listing by the numeric indicator <210> followed by the sequenceidentifier (e.g. <210>1, <210>2, etc). The length, type of sequence(DNA, RNA, etc.) and source organism for each nucleotide sequence areindicated by information provided in the numeric indicator fields <211>,<212> and <213>, respectively. Nucleotide sequences referred to in thespecification are identified by the indicator SEQ ID NO: followed by thesequence identifier (e.g. SEQ ID NO:1, SEQ ID NO:2, etc.). The sequenceidentifier referred to in the specification correlates to theinformation provided in numeric indicator field <400> in the sequencelisting, which is followed by the sequence identifier (e.g. <400>1,<400>2, etc.). That is, SEQ ID NO:1 as detailed in the specificationcorrelates to the sequence indicated as <400>1 in the sequence listing.

One aspect of the present invention is directed to a method of screeninga mammal for the onset or predisposition to the onset of aneuropsychiatric condition said method comprising determining thefunctional level of protein 14-3-3ζ in a biological sample derived fromsaid mammal wherein a lower level of said protein 14-3-3ζ relative tocontrol levels is indicative of the onset or predisposition to the onsetof said condition.

In another aspect there is provided a method of screening a human forthe onset or predisposition to the onset of a neuropsychiatric conditionsaid method comprising determining the functional level of protein14-3-3ζ in a biological sample derived from said human wherein a lowerlevel of said protein 14-3-3ζ relative to control levels is indicativeof the onset or predisposition to the onset of said condition.

In still another aspect is therefore provided a method of screening ahuman for the onset or predisposition to the onset of a conditioncharacterised by one or more symptoms characteristic of schizophreniasaid method comprising determining the functional level of protein14-3-3ζ in a biological sample derived from said human wherein a lowerlevel of said protein 14-3-3ζ relative to control levels is indicativeof the onset or predisposition to the onset of said condition.

In yet another aspect there is provided a method of screening a humanfor the onset or predisposition to the onset of schizophrenia, saidmethod comprising determining the functional level of protein 14-3-3ζ ina biological sample derived from said human wherein a lower level ofsaid protein 14-3-3ζ relative to control levels is indicative or theonset or predisposition to the onset of said schizophrenia.

The present invention therefore provides a method of diagnosing theonset of a neuropsychiatric condition in a human presenting with one ormore positive or negative symptoms or thought disorder said methodcomprising determining the functional level of protein 14-3-3ζ in abiological sample from said human wherein a lower level of said protein14-3-3ζ relative to control levels is indicative of the onset of saidcondition.

In still yet another aspect the present invention is directed to amethod of screening a mammal for the onset or predisposition to theonset of a neuropsychiatric condition said method comprising determiningthe level of expression of the gene encoding protein 14-3-3ζ in abiological sample derived from said mammal wherein a lower level ofexpression relative to control levels is indicative of the onset orpredisposition to the onset of said condition.

In a further aspect the present invention is directed to a method ofscreening a mammal for the onset or predisposition to the onset of aneuropsychiatric condition said method comprising determining the levelof protein 14-3-3ζ/DISC1 complex formation in a biological samplederived from said mammal wherein a lower level of complex formationrelative to control levels is indicative of the onset or predispositionto the onset of said condition.

Still another further aspect of the present invention is directed to amethod of monitoring the progression of a neuropsychiatric condition ina mammal diagnosed with the onset of said condition said methodcomprising-determining the functional level of protein 14-3-3ζ in abiological sample derived from said mammal wherein an equal or lowerlevel of protein 14-3-3ζ relative to a level preciously obtained forthat mammal is indicative of a poor prognosis and a higher level ofprotein 14-3-3ζ relative to a level previously obtained for that mammalis indicative of an improved prognosis.

Yet another aspect of the present invention is directed to a method ofmonitoring a patient determined to be predisposed to the onset of aneuropsychiatric condition said method comprising determining thefunctional level of protein 14-3-3ζ in a biological sample derived fromsaid mammal wherein an equal or lower level of protein 14-3-3ζ relativeto a level, previously obtained for that mammal is indicative of animproved prognosis.

Another aspect of the present invention is directed to a non-humanmammal deficient in functional protein 14-3-3ζ in which a gene encodingprotein 14-3-3ζ has been detected.

Yet another aspect of the present invention is directed to a method ofscreening for an agent which mimics protein 14-3-3ζ functionality or14-3-3ζ/DISC1 complex formation or otherwise improves the symptoms of aschizophrenic phenotype, said method comprising administering to a14-3-3ζ knockout non-human animal a putative modulation agent andscreening for altered phenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1: 14-3-3ζ-deficient mice demonstrate abnormal cognitive andbehavioural traits. (a) 14-3-3ζ^(062−/−) mice (open bars; n=11) havegreater exploratory behaviour at 5-30 weeks of age than 14-3-3ζ^(062+/+)littermates (filled bars; n=11) in an open field test. (b)14-3-3ζ^(062−/−) mice (open bars; n=12) spend more time than14-3-3ζ^(062+/+) mice (filled bars: n=12) in the open arm in an elevatedplus maze. (c) 14-3-3ζ^(062−/−) mice (open circles; n=12) have lowercapacity than 14-3-3ζ^(062−/−) mice (closed squares; n=12) for bothspatial learning (Day 1-6) and memory (M1 and M2) in a cross maze escapetask test. (d) Compared to 14-3-3ζ^(062+/+) mice (filled bars; n=11) the14-3-3ζ^(062−/−) mice (open bars; n=11) have reduced PP1 with a prepulse(PP) of 2, 4, 8 and 16 dB over the 70 dB baseline and an inter-stimulusinterval of 100 msec. The average (Avg) of data from all PP intensitiesis also shown. Data from male and female mice is pooled in all graphs.Error bars are presented as mean±SEM. *, p<0.05; **, p<0.01; ***,p<0.001.

FIG. 2. 14-3-3ζ is expressed in the pyramidal cells of Ammon's horn andgranule neurons of the dentate gyros. (a) (i) Schematic representationof a coronal section through a 14.5 dpc embryonic mouse brain depictingthe different regions of the hippocampus. V, ventricle; IZ, intermediatezone; VZ, ventricular zone. (ii) Schematic representation of a coronalsection through P0 mouse hippocampus. Neurons from the hippocampalprimordium originate from the ventricular neuroepithelium (light blue)and neuroepithelium adjacent to the fimbria (dark blue). The threesubfields containing the pyramidal neurons of the cornu ammonis (CA1-3)that compose Ammon's horn and its layers (so, stratum oriens; sp,stratum pyramidale; sl, stratum lucidum; sr, stratum radiatum) aredepicted in relation to positioning of granular neurons in the dentategyrus (DG). (b) (i-ii) 14-3-3ζ immunoreactivity was detected in theintermediate zone of the 14.5 dpc developing hippocampus. (iii-iv) AtP0, 14-3-3ζ-positive neurons are located in the pyramidal cell layer.(v) Higher magnification of the pyramidal neurons (asterisks) shows that14-3-3ζ has a punctate cytoplasmic localisation. (c) X-gal stainingshowing the endogenous expression of 14-3-3ζ in P0, P7 and adult14-3-3ζ^(062+/−) hippocampi. The high level of 14-3-3ζ-lacZ expressionis evident in pyramidal and granular neurons. (d) Hippocampal neuronalculture. (i) 14-3-3ζ staining with EB1 (red). (ii) MAP2 positive (green)hippocampal neurons. (iii) Overlay of 14-3-3ζ and MAP2 highlights theco-expression in MAP2 positive neurites (arrow), (e) 14-3-3ζ protein (27kDa) is expressed in Ammon's horn and dentate gyrus of the WT mice.Western blot of lysates from adult WT and 14-3-3ζ^(062−/−) mice wereimmunoblotted and probed with antibody to 14-3-3ζ (EBI). Anti-(3-actin(42 kDa) antibody was used as a loading control. Seale bars=100 μm(bi-iv; c: di-iii), 25 μm (bv).

FIG. 3: 14-3-3-ζ-deficient mice displayed lamination defects of thehippocampus. (a) Nissl staining shows the hippocampal development of WTand 14-3-3ζ^(062−/−) mice from 14.5 dpc until postnatal-day-56 (P56).Hippocampal cells are dispersed in the stratum pyramidale (sp) of the14-3-3ζ^(062−/−) mice (iv, vi, viii). Arrowheads highlight theduplicated layer of the hippocampal pyramidal neurons in stratumradiatum (sr). Asterisks highlight the ectopically positioned pyramidalcells in the stratum oriens (so). Arrows indicate the loosely arrangedgranule neurons in the dentate gyros. (b) Thy1-YFP transgene expressionintroduced in to the 14-3-3ζ⁰⁶² background revealed severedisorganization of hippocampal pyramidal neurons in 14-3-3ζ^(062−/−)mice. Blue, DAPI; green, Thy1 expression. (c) Coronal sections of thehippocampus obtained from P0 (i-iv) and P56 (v-vi) mice of the indicatedgenotype. The deeper stratum pyramidale is populated by NeuN-positivepyramidal cells in WT hippocampi (iii. yellow arrowheads) forming auniform mature zone from CA 1 to CA3. In 14-3-3ζ^(062−/−) hippocampi,the maturation zone was less uniform with some NeuN-positive maturepyramidal cells ectopically positioned in both the deeper zone (yellowarrowheads) and superficial zone (white arrowheads) of the stratumpyramidale in CA3. In P56 14-3-3ζ^(062−/−) mice, immunostaining For NeuNhighlighted pyramidal cells in the duplicated CA3 subfields indicatingthat ectopic cells achieved maturation (vi). Scale bars: 100 um.

FIG. 4: BrdU-pulse-chase analysis indicates neuronal migration defect in14-3-3ζ-deficient mice. BrdU-pulse-chase analysis at 14.5 dpc:P7 (i-v)and 16.5 dpc:P7 (vi-x) demonstrates that the BrdU-positive cells (black)locate within the stratum pyramidale (sp) in the CA3 subfield of WThippocampi (ii & vii). (v) Graph summarizes the percentage of theectopic hippocampal neurons at 14.5 dpc:P7. BrdU-labelled cells of14-3-3ζ^(062−/−) mice were ectopically positioned. Neurons were stalledin the stratum oriens (so), or migrated beyond the stratum pyramidaleand into the stratum lucidum (sl) (arrowheads in iv & ix). (x) Graphsummarizes the percentage of the ectopic hippocampal neurons at 16.5dpc:P7. Scale bars: 100 μm

FIG. 5: Abnormal mossy fibre pathways in 14-3-3ζ-deficient mice.Calbindin immunostaining of the infrapyramidal (IPMF, yellow arrowheads)and the suprapyramidal (SPMF, white arrowheads) mossy fibre trajectoriesin 14-3-3ζ^(062+/+) (i, iii, v and vii) and 14-3-3ζ^(062−/−) (ii, iv, viand viii) mice. Similar to WT controls, 14-3-3ζ^(062−/−) deficientneurites initially bifurcate into the SPMF and IPMF branches afternavigating away from the dentate gyrus (DG). However, the WWF branch of14-3-3ζ^(062−/−) mice navigated aberrantly among the pyramidal cellsomata (sp. whitearrows). In addition, the diffuse SPMF branch of14-3-3ζ^(062−/−) mice invaded the duplicated pyramidal cell-layer inCA3. Scale bars=100 μm.

FIG. 6: Functional synaptic connection between ectopic CA3 pyramidalcells and misrouted mossy fibres. (a) (i-iv) Hippocampal sections fromP56 14-3-3ζ^(062+/+) mice stained with antibodies to synaptophysin (Syp)show immunoreactivity in both the IPMF (white arrowheads) and SPMF(yellow arrowheads). Syp staining is located in both the stratum oriens(so) and stratum lucidum (sl), surrounding the pyramidal somata of CA3.(v-viii) Syp staining of hippocampal sections from 14-3-3ζ^(062−/−) micereveals that the mossy fibres navigating abnormally within the stratumpyramidale of CA3 (asterisks, v, vii) form functional synapses. (ix-xii)Ectopic mature CA3 pyramidal cells (stained by NeuN; depicted withasterisks) communicate with the synaptic protein (Syp, green) from themisrouted mossy fibres. Scale bars=100 μm. (b) Golgi stain reveals thedendritic arborization of the pyramidal cells of WT or 14-3-3ζ^(062−/−)adult mice (P35). A set of thorny excrescences, indicating the contactpoints with the misrouted mossy fibre synaptic boutons (MFB, bevelledline), is located on the apical proximal dendrites of CA3 pyramidalcells in WT neurons. Two sets of thorny excrescences are located on theapical dendritic tree in 14-3-3ζ^(062−/−) mice, one at the proximalapical dendrites and the other in the distal dendritic branches (*). (c)Schematic diagram depicts the misrouted mossy fibre trajectories andaberrant synaptic points of mossy fibre boutons communicating to theectopic CA3 pyramidal cells in 14-3-3ζ^(062−/−) mice as compared to WThippocampi.

FIG. 7: 14-3-3ζ interacts with DISC1 to control neuronal development.(a-b). Equal amounts of lysate from P7 mouse brains wereimmunoprecipitated with anti-DISC1 antibodies or anti-14-3-3 antibodiesand immunoblotted with DISC1 (a), or EB1 purified antisera to recognize14-3-3ζ (b). The relative expression levels of DISC1 isoforms and14-3-3ζ^(062−/−) from 5% of total cell lysate (input) used forco-immunoprecipitation were also determined by direct immunoblotting.Arrows indicate the major 100 kDa and 75 kDa bands of DISC1 (a) and 27kDa band representing 14-3-3ζ (b). Asterisk represents background IgGbands from immunoprecipitation. (c) Schematic representation of the roleof 14-3-3ζ in neuronal migration and axonal growth. (i) 14-3-3ζ bindsCDK5 phosphorylated Ndel1 to promote interaction with LIS1 and therebypromote neuronafmigration. (ii) is also present in the LIS1/Ndel1/DISC1complex to control axonal growth dynamics.

FIG. 8: Gene trap mutation of the 14-3-3ζ gene. (a) Schematic showingthe insertion point for mouse line 14-3-3ζ^(Gt(OST062)Lex) and (b) formouse line 14-3-3ζ^(Gt(OST390)Lex). The gene trap vector contains asplice acceptor sequence (SA) fused to a selectable marker gene (BGEOfor 0 galactosidase/neontycin phosphotransferase fusion gene) that isthereby expressed under the endogenous 14-3-3ζ promoter. When integratedinto the upstream exons of 14-3-3ζ BGEO produces a fusion transcriptthat interrupts mRNA transcription. The vectors also contain a PGKpromoter followed by the first exon of Bruton's Tyrosine Kinase gene(BTK) upstream of a splice donor (SD) signal. BTK contains terminationcodons in all reading frames to prevent translation of downstream fusiontranscripts. The gene trap vector is depicted in retrovirus form betweentwo long terminal repeats (LTR). On both figures, arrows denote primersused for genotyping, Red boxes indicate non-coding untranslated sequenceand green boxes denote coding sequence.

FIG. 9: Western Blot analysis demonstrates that 14-3-3ζ expression isreduced in all tissues of mutant mice: Tissue were harvested from (a)both male and female 14-3-3ζ^(062−/−) and age-matched 14-3-3ζ^(062+/+)mice and from (b) both male and female 14-3-3ζ^(390−/−) and age-matched14-3-3ζ^(390+/+) mice. All samples were homogenised in NP40 lysis buffercontaining protease inhibitors as described-in the Materials andMethods. Protein concentrations were determined using Pierce BCA ProteinAssay kit and 10 μg protein was loaded per lane. Blots were probed withEB-1 antibody to detect 14-3-3ζ and anti-β-actin (1:5000) was used as aloading control. Bound antibodies were detected with HRP-conjugatedsecondary antibody (1:20,000, Pierce-Thermo Scientific). Immunoreactiveproteins were visualized by ECL. Note that EB1 antibody may also detect14-3-3 isoforms other than 14-3-3ζ.

FIG. 10: mRNA levels of 14-3-3 isoforms remain constant in14-3-3ζ-deficient mouse brain: Transcript levels of all 14-3-3 isoformsare unchanged in response to the deletion of the 14-3-3ζ isoform inbrain tissue from 14-3-3ζ^(062−/−) mice. RNA was isolated from wholebrain of three 14-3-3ζ^(062−/−) mice and three age-matched14-3-3ζ^(062+/+) controls. Complementary DNA (cDNA) was generated from 1μg RNA using Quantitect kit (Qiagen). Real Time PCR using Sybr Green(Qiagen) and Rotor Gene machines (Corbett) was used to determine levelsof mRNA compared to GAPDH in samples for all isoforms of 14-3-3. SecTable 1 for primer details.

FIG. 11: 14-3-3ζ-deficient mice display cognitive dysfunction inlearning and memory. 14-3-3ζ^(062−/−) mice (open circles; n=12) havelower capacity than 14-3-3ζ^(062+/+) mice (closed squares; n=12) forboth spatial learning (Day 1-6) and memory in a cross maze escape tasktest. 14-3-3ζ^(062−1/−) mice take longer to reach the escape platformthroughout the training period and during the memory test: phase (M1 andM2). Data from male and female mice is pooled. Error bars are presentedas mean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 12: 14-3-3ζ-deficient mice display reduced startle reflex. Startleamplitude of 14-3-3ζ^(062−/−) mice (open bar; n=13) is lower than14-3-3ζ^(062+/+) mice (closed bars; n=14) over four pulse-alone blocksof 115 dB. The average (Avg) startle from all blocks is also shown. **,<0.05.

FIG. 13: 14-3-3ζ expression is maintained in hippocampal neurons. X-galstaining showing the endogenous expression of 14-3-3ζ in P0 and P714-3-3ζ^(062+/−) hippocampus and cerebellum. The high level of14-3-3ζ-lacZ expression in the hippocampus is evident in both thepyramidal neurons of the Ammon's horn and the mature dentate neurons butnot in the cerebellum post-birth. Scale bar=25 μm.

FIG. 14. Hippocampal lamination defects in 14-3-3ζ-deficient mice, Nisslstaining shows the hippocampal development of WT (i, iii, v) andt4-3-3ζ^(062−/−) (ii, iv, vi) mice from 14.5 dpc until birth (P0).Hippocampal cells were dispersed in the stratum pyramidale (sp) of the14-3-3ζ^(062−/−) mice. Arrowheads highlight the duplicated layer of thehippocampal pyramidal neurons in stratum radiatum (sr). Asteriskshighlight the ectopically positioned pyramidal cells in the stratumoriens (so). Scale bar=25 μm.

FIG. 15: Mispositioned neurons in 14-3-3ζ-deficient mice survive intoadulthood. Apoptotic cells in hippocampal primordium (a-f) and maturehippocampi (g-h). No increase in fragmented, apoptotic-cell nuclei (asshown in the green TUNEL positive cells in aii and bii) were detected14-3-3ζ^(−/−) hippocampi. Scale bar=100 μm.

FIG. 16: 14-3-3ζ controls glutamataregic pathways to mediatesensorimotor gating. Compared to 14-3-3ζ^(062+/+) mice (grey bar; n=11;5 male and 6 female) the 14-3-3ζ^(062−/−) mice (grey hashed bar: n=11; 5male and 6 female) have reduced PPI with a prepulse (PP) of 16 d11 overthe 70 dB baseline and an inter-stimulus interval of 100 msec. BothMK801 (MK) and Apomorphine (APO) induce PPI defects in 14-3-3ζ^(062+/+)mice. In contrast, only APO induces further PPI defects in14-3-3ζ^(062−/−) mice.

FIG. 17: 14-3-3ζ controls dopaminergic pathways to mediate locomotoractivity. 14-3-3ζ^(062−/−) mice (open bars; n=11; 6 male and 5 female)have greater exploratory behaviour at 30 weeks of age than14-3-3ζ^(062+/+) littermates in an open field test when treated withD-Amphetamine. Sal, saline. Data from male and female mice is pooled inall graphs. Error bars arc presented as mean=SEM. *, p<0.05.

FIG. 18: 14-3-3ζ-deficient mice have reduced spine density.

(A) Dendritic spines are reduced in the granular neurons of the dentategyrus (DC) in 14-3-3ζ^(062−/−) mice (n=3, 20 dendrites counted from eachanimal) compared to 14-3-3ζ^(062+/+) mice (n+4, 20 dendrites countedfrom each animal). (B) Dendritic spines are also reduced in thepyramidal neurons of the cornu ammonis (CA) in 14-3-3ζ^(062−/−) mice(n=3, 20 dendrites counted from each animal) compared to14-3-3ζ^(062+/+) mice (n+4, 20 dendrites counted from each animal); Leftpanels show representative images of spines in a 14-3-3ζ^(062−/−) and14-3-3ζ^(062+/+) DG dendrites or CA dendrites. Right panels indicatequantification with relevant p values. Note, scoring of spine numberswas completed blind for all animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thata reduction in the functional level of protein 14-3-3ζ, such as in thecontext of absolute levels of protein 14-3-3ζ or levels of protein14-3-3ζ/DISC1 complex formation, is indicative of the onset orpredisposition to the onset of a neuropsychiatric condition, such asschizophrenia or related condition. This finding has thereforefacilitated the development of a simple yet highly useful test forassessing susceptibility to or diagnosing onset of a neuropsychiatriccondition: Also facilitated is the generation of genetically modifiedanimals which do not express protein 14-3-3ζ and methods for their use,such as in the context of developing or testing known or new therapeuticor prophylactic treatment regimes or screening for modulatory agentswhich may be useful in this regard.

Accordingly, one aspect of the present invention is directed to a methodof screening a mammal for the onset or predisposition to the onset of aneuropsychiatric condition said method comprising determining thefunctional level of protein 14-3-3ζ in a biological sample derived fromsaid mammal wherein a lower level of said protein 14-3-3ζ relative tocontrol levels is indicative of the onset or predisposition to the onsetof said condition.

Without limiting the present invention to any one theory or mode ofaction, the 14-3-3 proteins are a conserved family of dimericphospho-serine binding proteins that interact and modulate the functionsof multiple celiniar proteins and in so doing regulate many signallingpathways (Tzivion and Avruch 2002, J. Biol. Chem. 277:3061-64; Fu et al.2000, Ann. Rev. Pharma. Tax. 40:617-47). The 14-3-3 proteins arecomposed of two 30 kDa monomer units that are each capable of binding aphospho-serine motif via an amphipathic groove. Dimers of 14-3-3 areformed by the N-terminal α helices, with helix 1 of one monomerinteracting with helices 3 and 4 of another. Functionally, 14-3-3proteins perform multiple roles in regulating cellular proteinactivities and importantly, these functions of 14-3-3 are dependent onits dimeric structure (Xing et al. 2000, supra; Yaffe 2002, FEBS Letts.513:53-57). The 14-3-3 proteins are a highly conserved family of sevenphospho-serine binding proteins. The seven isoforms are the β, ε, γ, η,σ, τ and ζ (NCBI Ref. Sequence Number NM_003405.3; SEQ ID NO:1) forms.

Reference to “protein 14-3-3ζ” should be understood to include referenceto all forms of protein 14-3-3ζ including functional allelic orpolymorphic variants. Reference to “variants” should be understood toextend to functional mutants. Reference to “homologues” should beunderstood as a reference to 14-3-3 proteins from species other thanhuman. Reference to a “functional” 14-3-3 protein should be understoodas a reference to a molecule which can undergo DISC1 complex formationand thereby facilitate ongoing signalling via the DISC1 regulatednetwork. It should he understood that “protein 14-3-3ζ” is alsointerchangeably referred to as “14-3-3ζ” in this specification. Bothterms should be understood as a reference to the same molecule. In oneembodiment, said protein 14-3-3ζ is human 14-3-3ζ.

According to this embodiment there is provided a method of screening ahuman for the onset or predisposition to the onset of a neuropsychiatriccondition said method comprising determining the functional level ofprotein 14-3-3ζ in a biological sample derived from said human wherein alower level of said protein 14-3-3ζ relative to control levels isindicative of the onset or predisposition to the onset of saidcondition.

Reference to a “neuropsychiatric condition” should be understood as areference to a condition characterised by neurologically basedcognitive, emotional and behavioural disturbances. Examples of suchconditions include, inter alia, a condition characterised by one or moresymptoms of schizophrenia, schizophrenia, schizotypal personalitydisorder, psychosis, bipolar disorder, manic depression, affectivedisorder, or schizophreniform or schizoaffective disorders, psychoticdepression, autism, drug induced psychosis, delirium, alcohol withdrawalsyndrome or dementia induced psychosis.

In one embodiment, said neuropsychiatric condition is a condition whichis characterised by one or more symptoms of schizophrenia.

According to this embodiment there is therefore provided a method ofscreening a human for the onset or predisposition to the onset of acondition characterised by one or more symptoms characteristic ofschizophrenia said method comprising determining the functional level ofprotein 14-3-3ζ in a biological sample derived from said human wherein alower level of said protein 14-3-3ζ relative to control levels isindicative of the onset or predisposition to the onset of saidcondition.

Reference to “symptoms characteristic of schizophrenia” should beunderstood as a reference to any one or more symptoms which may occur inan individual suffering from schizophrenia. These symptoms may beevident throughout the disease course or they may be evident onlytransiently or periodically. For example, the hallucinations associatedwith schizophrenia usually occur in periodic episodes while thecharacteristic social withdrawal may exhibit an ongoing manifestation.It should also be understood that the subject symptoms may notnecessarily be exhibited by all individuals suffering fromschizophrenia. For example, some individuals may suffer from auditoryhallucinations only while others may suffer only from visualhallucinations. However, for the purpose of the present invention, anysuch symptoms, irrespective of how many or few schizophrenia patientsever actually exhibit the given symptom, are encompassed by thisdefinition. Without limiting the present invention to any one theory ormode of action, the symptoms that are most commonly associated with thedisease are called positive symptoms (which denote the presence ofgrossly abnormal behaviour), thought disorder and negative symptoms.Thought disorder and positive symptoms include speech which is difficultto follow or jumping from one subject to another with no logicalconnection, delusions (false beliefs of persecution, guilt, grandeur orbeing under outside control) and hallucinations (visual or auditory).Thought disorder is the diminished ability to think clearly andlogically. Often it is manifested by disconnected and nonsensicallanguage that renders the person with schizophrenia incapable ofparticipating in conversation, contributing to alienation from family,friends and society. Delusions are common among individuals withschizophrenia. An affected person may believe that he or she is beingconspired against (called “paranoid delusion”). “Broadcasting” describesa type of delusion in which the individual with this illness believesthat their thoughts can be heard by others. Hallucinations can be heard,seen or even felt. Most often they take the form of voices heard only bythe afflicted person. Such voices may describe the person's actions,warn of danger or tell him what to do. At times the individual may hearseveral voices carrying on a conversation less obvious than the“positive symptoms” but equally serious are the deficit or negativesymptoms that represent the absence of normal behaviour. These includeflat or blunted affect (i.e. lack of emotional expression), apathy,social withdrawal and lack of insight. Both the positive symptoms andthe negative symptoms should be understood to fall within the definitionof “symptoms characteristic of schizophrenia”.

In addition to the fact that there may be significant variation betweenschizophrenia patients in terms of which symptoms they exhibit, itshould also be understood that there are other neuropsychiatricconditions which are also characterised by one or more of thesesymptoms. Hallucinations, for example, are also commonly observed inpatients with bipolar disorder, psychotic depression, delirium anddementia induced psychosis, for example. Accordingly, reference to acondition characterised by one or more symptoms characteristic ofschizophrenia should be understood as a reference to anyneuropsychiatric condition which is characterised by the presence of oneor more of these symptoms. In one embodiment, said condition isschizophrenia.

According to this embodiment there is provided a method of screening ahuman for the onset or predisposition to the onset of schizophrenia,said method comprising determining the functional level of protein14-3-3ζ in a biological sample derived from said human wherein a lowerlevel of said protein 14-3-3ζ relative to control levels is indicativeof the onset or predisposition to the onset of said schizophrenia.

The term “mammal” as used herein includes humans, primates, livestockanimals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory testanimals (e.g. mice, rats, guinea pigs), companion animals (e.g. dogs,cats) and captive wild animals (e.g. kangaroos, deer, foxes).Preferably, the mammal is a human or a laboratory test animal. Even morepreferably, the mammal is a human.

Individuals exhibiting protein 14-3-3ζ levels lower than the normalrange are generally regarded as having undergone the onset of thesubject condition, as opposed to a predisposition to its onset, wherethere is also evidence of the onset of one or more positive or negativesymptoms, as hereinbefore described. It would be appreciated that one ofthe limitations of diagnosing neuropsychiatric conditions such asschizophrenia has been that in the early stages of the condition, thesymptoms which are observed are non-specific and can often be attributedto non-neuropsychiatric causes. This makes diagnosis extremelydifficult, particularly when the stigma associated with having acondition such as schizophrenia can lead to a reluctance by healthprofessionals or patients' families to acknowledge such a diagnosis inthe absence of absolute certainty in this regard. Where an incorrectdiagnosis is made, the consequences can be serious since a therapeutictreatment regime may be designed which will not, in fact, be effectiveor may even be detrimental. Accordingly, the method of the presentinvention now provides a means of obtaining a conclusive diagnosis inthese types of situations since the observance of the onset of symptomscharacteristic of a neuropsychiatric condition can now be assessedtogether with a biochemical readout in order to provide a firmdiagnosis.

The present invention therefore provides a method of diagnosing theonset of a neuropsychiatric condition in a human presenting with one ormore positive or negative symptoms or thought disorder said methodcomprising determining the functional level of protein 14-3-3ζ in abiological sample from said human wherein a lower level of said protein14-3-3ζ relative to control levels is indicative of the onset of saidcondition.

In one embodiment, said condition is a condition characterised by one ormore symptoms of schizophrenia.

In another embodiment, said condition is schizophrenia.

As detailed hereinbefore, the present invention also provides a means ofdetermining whether or not a patient is predisposed to developing aneuropsychiatric condition. In this situation, the individual istypically not yet exhibiting any symptoms of a neuropsychiatriccondition. However, due to any one of a number of factors, such as afamily history for example, it may be desirable to determine whetherthat individual is predisposed to the onset of such a condition. Thiscan provide valuable information which may enable lifestyle changes tobe made or prophylactic drug treatment regimens to be implemented.Reference to “predisposition” in this regard should therefore beunderstood as a reference to an increased tendency or susceptibility tothe onset of such condition relative to a normal individual. It does notmean that the individual will necessarily develop the condition, forexample if the individual is not exposed to a trigger for the onset ofthe condition, but merely that under circumstances where the onset ofthe condition could be triggered, it is more likely that a personpredisposed to the condition would develop it than someone who is not.Reference to an individual who is “not predisposed” to the onset of sucha condition should be understood to have the converse meaning. Referenceto “predisposition” is also intended to describe an individual whoexhibits an increased risk of developing a neuropsychiatric conditionrelative to that of the general population.

The present invention is predicated on the determination that areduction in the functional level of protein 14-3-3ζ is indicative ofthe onset or predisposition to the onset of a neuropsychiatriccondition. Reference to the “functional level” should be understood as areference to the biologically effective level of protein 14-3-3ζ ratherthan its absolute level, per se. That is, the capacity of protein14-3-3ζ to effect the biological pathways hereinafter described is therelevant issue, this being impacted upon by measurable factors otherthan just absolute levels of protein 14-3-3ζ.

Without limiting the present invention to any one theory or mode ofaction, 14-3-3ζ binds to phosphorylated Ndel1 , maintainsphosphorylation of Ndel1 and thereby promotes Ndel1 binding to LIS1 andcytoplasmic dynein heavy chain that regulate nuclear movement. However,it has also now been determined that protein 14-3-3ζ interacts with thetripartite DISC1/Ndel1 /LIS1-complex to promote kinesin motor bindingand relocation of Ndel1/LIS1 to growing axons. Still further, thispreferentially occurs in an isoform specific manner, with the14-3-3ζ/DISC1 complex formation and signalling preferentially occurringin the context of the 75 kDa isoform of DISC1 rather than the 100 kDaisoform. These findings are in contrast to the previous findings whichdemonstrated that Ndel1 in fact interacted with the 100 kDa isoform ofDISC1.

The DISC1 protein is encoded by the DISC1 gene in humans (Millar et at.2000, Hum. Mol. Genet. 9(9): 1415-23). In coordination with a wide arrayof interacting partners, DISC1 has been-shown to participate in theregulation of cell proliferation, differentiation, migration, neuronalaxon and dendrite outgrowth, mitochondrial transport, fission and/orfusion, arid cell-to-cell adhesion. The DISC1 gene is situated atchromosome 1q42.1 and overlaps with DISC2 open reading frame. MultipleDISC1 isoforms have been identified at the RNA level, including aTSNAX-DISC1 transgene splice variant, and at the protein level (Nakataet al. (2009). Proc Natl Acad Sci USA. 106(37):15873-8). Of the isolatedRNA isomers, 4 have been confirmed to be translated namely Long form(L), Long variant isoform (Lv), Small isoform (S), and Especially smallisoform (Es), Human DISC1 is transcribed as two major splice variants. Lform and Lv isoform. The L and Lv transcripts utilize distal andproximal splice sites, respectively, within exon 11. Alternatetranscriptional splice variants, encoding different isoforms, have beencharacterized. DISC1 homologues have been identified in the commonchimpanzee, the Rhesus monkey, the house mouse, the brown rat,zebrafish, pufferfish, cattle, and dogs (Taylor et al. (2003) Genomics81(1):67-77).

The protein encoded by this gene, is predicted to contain a coiled coilmotif rich C-terminal domain and a N-terminal globular domain (Taylor etal. 2003 supra). The N-terminus contains two putative nuclearlocalization signals and a serine-phenylalanine-rich motif of unknownsignificance. The C-terminus contains multiple regions with coiled-coilforming potential and two leucine zippers that may mediateprotein-protein interactions. The DISC1 protein has no known enzymaticactivity; rather it exerts its effect on multiple proteins throughinteractions to modulate their functional states and biologicalactivities in time and space (Bradshaw and Porteous (2010-12-31).“DISC1-binding proteins in neural development, signalling andschizophrenia.”Neuropharmacology).

Reference to “DISC1” should be understood to have a correspondingmeaning to that provided in relation to “protein 14-3-3ζ”.

Still without limiting the present invention in any way, a reduction inprotein 14-3-3ζ functional levels, in the context of a schizophreniamouse model, induced behavioural and cognitive defects characteristic ofschizophrenia. These deficiencies were noticed as an increase inlocomotor function, inability to recognise novel objects, reducedanxiety to an open environment, severely reduced capacity to learn orremember and abnormal sensorimotor gating. Anatomical disturbances ofneurons within the hippocampus were also identified and are postulatedto arise from aberrant neuronal migration. Also identified were specificaxonal navigation defects and abnormal synaptic connectivity ofhippocampal mossy fibres, this being consistent with schizophrenia beinga disorder of the synapse. More specifically, laminar organisation ofthe hippocampus was disrupted where 14-3-3ζ functionality was adverselyimpacted upon. This defect arose primarily from aberrant migration ofhippocampal neurons from the subventricular zone to their usual restingplace in the stratum pyramidale. The 14-3-3ζ/DISC1 axis is therefore acentral biological pathway in the pathophysiology of schizophrenia andthe observed anatomical defects are consistent with the fact that thecognitive defects associated with schizophrenia arise from bothneurodevelopmental deficiencies and disturbances of the synapse.

In terms of assessing the “biologically effective level” of protein14-3-3ζ functionality, it should be understood that this can be assessednot only in terms of absolute levels of protein 14-3-3ζ, but also at thelevel of protein 14-3-3ζ/DISC1 complex formation since it is actuallythe formation of these complexes which underpins the requisiteneurological development. Defects in absolute levels of protein 14-3-3ζwill impact on this complex formation but so too will other structuralor functional defects in either the protein 14-3-3ζ molecule or theDISC1 molecule since these lead to an inability for effective complexformation to occur. Without complex formation, the downstream signallingevents which are induced by this complex cannot occur. Accordingly, onecan therefore screen for both defects in protein 14-3-3ζ expression anddefects of other types which may not necessarily impact on protein14-3-3ζ levels but which nevertheless adversely impact on the ability of14-3-3ζ to form functional complexes with DISC1. For example, one canscreen for:

-   (i) a decrease in the protein 14-3-3ζ translation product level;-   (ii) a decrease in the protein 14-3-3ζ transcription product level    (for example, primary RNA or mRNA);-   (iii) changes to the chromatin proteins with which the 14-3-3 gene    is associated, for example the presence of histone H3 methylated on    lysine at amino acid position number 9 or 27 (repressive    modifications) or changes to the DNA itself which act to    downregulate expression, such as changes to the methylation of the    DNA;-   (iv) a reduction in the ability of protein 14-3-3ζ and DISC1 to form    complexes.

In accordance with this embodiment, the present invention is directed toa method of screening a mammal for the onset or predisposition to theonset of a neuropsychiatric condition said method comprising determiningthe level of expression of the gene encoding protein 14-3-3ζ in abiological sample derived from said mammal wherein a lower level ofexpression relative to control levels is indicative of the onset orpredisposition to the onset of said condition.

It should be understood that reference to the “expression” of theprotein 14-3-3ζ gene is a reference to assessing the level of either thetranscription product encoding this particular protein 14-3-3 isoform(e.g. mRNA) or the translation protein (i.e. the protein) itself.

In another embodiment, said level of expression is mRNA expression.

In yet another embodiment, said level of expression is proteinexpression.

In still another embodiment, said level of expression is assessed byscreening for changes to genomic DNA, such as changes to DNAmethylation, in particular hypermethylation.

In yet still another embodiment, said level of expression is assessed byscreening for changes to the chromatin protein with which said gene isassociated.

In a further embodiment the present invention is directed to a method ofscreening a mammal for the onset or predisposition to the onset of aneuropsychiatric condition said method comprising determining the levelof protein 14-3-3ζ/DISC1 complex formation in a biological samplederived from said mammal wherein a lower level of complex formationrelative to control levels is indicative of the onset or predispositionto the onset of said condition.

In one embodiment, said complex is a complex between protein 14-3-3ζ andthe 75 kDa DISC1 isoform.

It should he understood by the person of skill in the art that referenceto the “protein 14-3-3ζ/DISC1 complex” is a reference to a complexcomprising both protein 14-3-3ζ and DISC1 but not necessarily only thosetwo molecules. That is, the complex may include other molecules, such asNdel1 and LIS1. It should also be understood that the 14-3-3ζ proteinmay not necessarily interact directly with or exclusively to DISC1. Thatis, it may also interact with Ndel1 and/or LIS1.

The method of the present invention is predicated on the analysis of thelevel of protein 14-3-3ζ expression in a biological sample relative to acontrol level of this marker. The “control level” may be either a“normal level”, which is the level of protein 14-3-3ζ expression in acorresponding sample taken from an individual who is not suffering fromthe subject neuropsychiatric condition, or it may be a sample harvestedat an earlier point in time from the patient in issue. The lattersituation is relevant where the method of the invention is used tomonitor a patient over a period of time, for example to assess theeffectiveness ore therapeutic or prophylactic treatment regime. This isdiscussed in more detail hereafter, it should also be understood thatthe subject protein 14-3-3ζ may be assessed or monitored by eitherquantitative or qualitative readouts.

The normal level may be determined using a biological samplecorresponding to the sample being analysed but which has been isolatedfrom an individual who has not developed the condition nor ispredisposed to developing the condition. However, it would beappreciated that it is likely to be most convenient to analyse the testresults relative to a standard result which reflects individual orcollective results obtained from healthy individuals. This latter formof analysis is in fact the preferred method of analysis since it enablesthe design of kits which require the collection and analysis of a singlebiological sample being a test sample of interest. The standard resultswhich provide the normal level may be calculated by arty suitable meanswhich would be well known to the person of skill in the art. Forexample, a population of normal tissues can be assessed in terms of thelevel of 14-3-3ζ thereby providing a standard value or range of valuesagainst which all future test samples are analysed. It should also beunderstood that the normal level may be determined from the subjects ofa specific cohort and for use with respect to test samples derived fromthat cohort. Accordingly, there may be determined a number of standardvalues or ranges which correspond to cohorts which differ in respect ofcharacteristics such as age, gender, ethnicity or health status. Said“normal level” may be a discrete level or a range of levels.

Although the preferred method is to detect a decrease in protein 14-3-3ζlevels order to diagnose the onset of or predisposition to the onset ofthe subject condition, the detection of increases in protein 14-3-3ζlevels may be desired under certain circumstances. For example, one mayseek to monitor an individual for changes in disease state or forprognostic implications in relation to the development or onset of acuteepisodes of psychosis, such as during the course of prophylactic ortherapeutic treatment of the patients. Alternatively, patientspresenting with symptoms of schizophrenia, for example, or a genetic orenvironmental predisposition to the development of such a condition maybe monitored. It should be understood that in accordance with thisaspect of the present invention, 14-3-3ζ functional protein levels willlikely be assessed relative to one or more previously obtained results,as hereinbefore described.

The method of the present, invention is therefore useful as a one offtest, as an on-going monitor of those individuals thought to be at riskof the development of such a condition or as a monitor of theeffectiveness of therapeutic or prophylactic treatment regimes directedto inhibiting or otherwise slowing the onset or progression of such acondition. Accordingly, the method of the present invention should beunderstood to extend to monitoring far increases or decreases in protein14-3-3ζ levels in an individual relative to a normal level (ashereinbefore defined) or relative to one or more earlier levelsdetermined from said individual.

Accordingly, another aspect of the present invention is directed to amethod of monitoring the progression of a neuropsychiatric condition ina mammal diagnosed with the onset of said condition said methodcomprising determining the functional level of protein 14-3-3ζ in abiological sample derived from said mammal wherein an equal or lowerlevel of protein 14-3-3ζ relative to a level previously obtained forthat mammal is indicative of a poor prognosis and a higher level ofprotein 14-3-3ζ relative to a level previously obtained for that mammalis indicative of an improved prognosis.

Yet another aspect of the present invention is directed to a method ofmonitoring a patient determined to be predisposed to the onset of aneuropsychiatric condition said method comprising determining thefunctional level of protein 14-3-3ζ in a biological sample derived fromsaid mammal wherein an equal or lower level of protein 14-3-3ζ relativeto a level previously obtained for that mammal is indicative of animproved prognosis.

In one embodiment, said condition is characterised by one or moresymptoms of schizophrenia.

In another embodiment, said condition is schizophrenia.

In still another embodiment, said mammal is a human.

In another embodiment, said level of expression is mRNA expression.

In yet another embodiment, said level of expression is proteinexpression.

In still another embodiment, said level of expression is assessed byscreening for changes to genomic DNA, such as changes to DNAmethylation, in particular hypermethylation.

In yet still another embodiment, said level of expression is assessed byscreening for changes to the chromatin protein with which said gene isassociated.

In still yet another embodiment said functional level is the level ofprotein 14-3-3ζ/DISC1 complex formation, more particularly 14-3-3ζ/75kDa DISC1 isoform complex formation.

Without limiting the present invention in any way, the method of thepresent invention is particularly useful since protein 14-3-3ζexpression is detectable outside the brain. Most previously identifiedmarkers of schizophrenia have taken the form of mutations present in thetranscription product of genes expressed only in the brain. From adiagnostic perspective, this is not desirable since the prospect ofharvesting brain tissue for testing, particularly routine testing, isunpalatable to the patient and potentially open to the development ofserious complications, such as infection, due to its highly invasivenature. The present findings, however, have enabled the use of otherbiological sources, including the cerebrospinal fluid, peripheral bloodand adult derived neural stem cells from tissues such as dental pulp,hair follicle and nasal pit. The development of this test has nowrendered possible significantly simpler and routine testing ofindividuals for schizophrenia.

Reference to a “biological sample” should therefore be understood as areference to any sample of biological material derived from an animalsuch as, but not limited to, cellular material, tissue biopsy specimensor bodily fluid (e.g. cerebrospinal fluid or blood). The biologicalsample which is tested according to the method of the present inventionmay be tested directly or may require some form of treatment prior totesting. For example, a biopsy sample may require homogenisation priorto testing or it may require sectioning for in situ testing. Further, tothe extent that the biological sample is not in liquid form, (if suchform is required for testing) it may require the addition of a reagent,such as a buffer, to mobilise the sample. Preferably, said biologicalsample is a sample of peripheral blood lymphocytes or adult derivedneural stem cells.

To the extent that the target molecule is present in a biologicalsample, the biological sample may be directly tested or else all or someof the nucleic acid material or protein present in the biological samplemay be isolated prior to testing. In yet another example the sample maybe partially purified or otherwise enriched prior to analysis. Forexample, to the extent that a biological sample comprises a very diversecell population, it may be desirable to select out a sub-population ofparticular interest (e.g. CNS cells) if mRNA is the subject of analysis.It is within the scope of the present invention for the target nucleicacid or protein molecule to be pre-treated prior to testing, for exampleinactivation of live virus or being run on a gel. It should also beunderstood that the biological sample may be freshly harvested or it mayhave been stored (for example by freezing) prior to testing or otherwisetreated prior to testing (such as by undergoing culturing).

The choice of what type of sample is most suitable for testing maccordance with the method disclosed herein will be dependent on thenature of the situation.

As detailed hereinbefore reference to “expression” should be understoodas a reference to the transcription and/or translation of a nucleic acidmolecule. Reference to “RNA” should be understood to encompass referenceto any form of RNA, such as primary RNA or mRNA. Without limiting thepresent invention in any way, the modulation of gene transcriptionleading to increased or decreased RNA synthesis will also correlate withthe translation of these RNA transcripts (such as mRNA) to a proteinproduct. Accordingly, the present invention also extends to detectionmethodology which is directed to screening for modulated levels orpatterns of the protein 14-3-3ζ products as an indicator of theneoplastic state of a cell or cellular population. Although one methodis to screen for mRNA transcripts and/or the corresponding proteinproduct, it should he understood that the present invention is notlimited in this regard and extends to screening for any other form ofexpression product such as, for example, a primary RNA transcript.

In terms of screening for the downregulation of expression of protein14-3-3ζ it would also be well known to the person of skill in the artthat changes which are detectable at the DNA level are indicative ofchanges to gene expression activity and therefore changes to expressionproduct levels. Such changes include but are not limited to, changes toDNA methylation. Accordingly, reference herein to “screening the levelof expression” and comparison of these “levels of expression” to control“levels of expression” should be understood as a reference to assessingDNA factors which are related to transcription, such as gene/DNAmethylation patterns.

It would also be known to a person skilled in the art that changes inthe structure of chromatin are indicative of changes in gene expression.Silencing of gene expression is often associated with modification ofchromatin proteins, methylation of lysines at either or both positions 9and 27 of histone H3 being well studied examples, while active chromatinis marked by acetylation of lysine 9 of histone H3. Thus association ofgene sequences with chromatin carrying repressive or activemodifications can be used to make an assessment of the expression levelof a gene.

Reference to “nucleic acid molecule” should be understood as a referenceto both deoxyribonucleic acid molecules and ribonucleic acid moleculesand fragments thereof. The present invention therefore extends to bothdirectly screening for mRNA levels in a biological sample or screeningfor the complementary cDNA which has been reverse-transcribed from anmRNA population of interest. It is well within the skill of the personof skill in the art to design methodology directed to screening foreither DNA or RNA. As detailed above, the method of the presentinvention also extends to screening for the protein product translatedfrom the subject mRNA or the genomic DNA itself.

In one embodiment, the level of protein 14-3-3ζ expression is measuredby reference to the mRNA or protein product.

In another embodiment, said gene expression is assessed by analysinggenomic DNA methylation. In another embodiment, expression is assessedby the association of DNA with chromatin proteins carrying repressivemodifications, for example, methylation of lysines 9 or 27 of histoneH3.

The term “protein” should be understood to encompass peptides,polypeptides and proteins (including protein fragments). The protein maybe glycosylated or unglycosylated and/or may contain a range of othermolecules fused, linked, bound or otherwise associated to the proteinsuch as amino acids, lipids, carbohydrates or other peptides,polypeptides or proteins. Reference herein to a “protein” includes aprotein comprising a sequence of amino acids as well as a proteinassociated with other molecules such as amino acids, lipids,carbohydrates or other peptides, polypeptides or proteins.

Reference to a “fragment” should be understood as a reference to aportion of the subject nucleic acid molecule or protein. This isparticularly relevant with respect to screening for modulated RNA levelssince these are inherently unstable molecules and may be screened for insamples which express high levels of enzymes. In this case the subjectRNA is likely to have been degraded or otherwise fragmented. One maytherefore actually be detecting fragments of the subject RNA molecule,which fragments are identified by virtue of the use of a suitablyspecific probe.

Means of assessing protein 14-3-3ζ in a biological sample can beachieved by any suitable method, which would be well known to the personof skill in the art. To this end, it would be appreciated that to theextent that one is examining either a homogeneous cellular population ora tissue section, one may utilise a wide range or techniques such as insitu hybridisation, assessment of expression profiles by microassays,immunoassays and the like (hereinafter described in more detail) todetect the absence of or downregulation of the level of expression ofone or more markers of interest. However, to the extent that one isscreening a heterogenous cellular population or a bodily fluid in whichheterogeneous populations of cells are found, such as a blood sample,the absence of or reduction in level of expression of protein 14-3-3ζ bya specific cellular subpopulation may be more difficult to detect due toinherent expression of protein 14-3-3ζ by other subpopulations of cellswhich are also present in the sample. That is, a decrease in the levelof expression of a subgroup of cells may not be detectable. In thissituation, a more appropriate mechanism of detecting a reduction in theexpression levels of protein 14-3-3ζ is via indirect means, such as thedetection of epigenetic changes.

As detailed hereinbefore, during development gene expression isregulated by processes that alter the availability of genes forexpression in different cell lineages without any alteration in genesequence, and these states can be inherited through a cell division aprocess called epigenetic inheritance. Epigenetic inheritance isdetermined by a combination of DNA methylation (modification of cytosineto give 5-methyl cytosine, 5 meC) and by modifications of the histonechromosomal proteins that package DNA. Thus methylation of DNA at CpGsites and modifications such as deacetylation of histone H3 on lysine 9,and methylation on lysine 9 or 27 are associated with inactivechromatin, while the converse state of a lack of DNA methylation,acetylation of lysine 9 of histone H3 is associated with open chromatinand active gene expression.

A variety of methods are available for detection of aberrantlymethylated DNA of a specific gene, even in the presence of a largeexcess of normal DNA (Clark 2007). Thus, loss of expression of a genewhich may be difficult to detect at the protein or RNA level except byimmunohistochemistry can often be detected by the presence ofhypermethylated DNA of the gene's promoter. Epigenetic alterations andchromatin changes arc also evident in the altered association ofmodified histones with specific genes (Esteller. 2007); for examplerepressed genes are often found associated with histone H3 that isdeacetylated and methylated on lysine 9. The use of antibodies targetedto altered histones allows for the isolation of DNA associated withparticular chromatin states and its potential use in cancer diagnosis.

Other methods of detecting changes to gene expression levels include butare not limited to:

(i) In Vivo Detection.

Molecular imaging may be used following administration of imaging probesor reagents capable of disclosing altered expression of protein 14-3-3ζ.Molecular imaging (Moore et al., BBA, 1402:239-249, 1988; Weissleder etal., Nature Medicine 6:351-355, 2000) is the in vivo imaging ofmolecular expression that correlates with the macro-features currentlyvisualized using “classical” diagnostic imaging techniques such asX-Ray, computed tomography (CT), MRI, Positron Emission Tomography (PET)or endoscopy.(ii) RNA ScreeningDetection of downregulation of RNA expression in cells by Fluorescent InSitu Hybridization (FISH), or in extracts from the cells by technologiessuch as Quantitative Reverse Transcriptase Polymerase Chain Reaction(QRTPCR) or Flow cytometric qualification of competitive RT-PCR products(Wederneyer et al., Clinical Chemistry 48:9 1398-1405, 2002),(iii) Assessment of Expression Profiles of RNA, for Example by ArrayTechnologies (Alon et al., Proc. Natl. Acad. Set USA: 96, 6745-6750,June 1999).

A “microarray” is a linear or multi-dimensional array of preferablydiscrete regions, each having a defined area, formed on the surface of asolid support. The density of the discrete regions on a microarray isdetermined by the total numbers of target polynucleotides to be detectedon the surface of a single solid phase support. As used herein, a DNAmicroarray is an array of oligonucleotide probes placed onto a chip orother surfaces used to amplify or clone target polynucleotides. Sincethe position of each particular group of probes in the array is known,the identities of the target polynucleotides can be determined based ontheir binding to a particular position in the microarray.

Recent developments in DNA microarray technology make it possible toconduct a large scale assay of a plurality of target nucleic acidmolecules on a single solid phase support. U.S. Pat. No. 5,837,832 (Cheeet al.) and related patent applications describe immobilizing an arrayof oligonucleotide probes for hybridization and detection of specificnucleic acid sequences in a sample. Target polynucleotides of interestisolated from a tissue of interest are hybridized to the DNA chip andthe specific sequences detected based on the target polynucleotidespreference and degree of hybridization at discrete probe locations. Oneimportant se of arrays is in the analysis of differential geneexpression, where the profile of expression of genes in different cellsor tissues, often a tissue of interest and a control tissue, is comparedand any differences in gene expression among the respective tissues areidentified. Such information is useful for the identification of thetypes of genes expressed in a particular tissue type and diagnosis ofconditions based on the expression profile.

The arrays may be produced according to any convenient methodology, suchas preforming the polynucleotide microarray elements and then stablyassociating them with the surface. Alternatively, the oligonucleotidesmay be synthesized on the surface, as is known in the art. A number ofdifferent array configurations and methods for their production areknown to those of skill in the art and disclosed in WO 95/25116 and WO95/35505 (photolithographic techniques), U.S. Pat. No. 5,445,934 (insitu synthesis by photolithography), U.S. Pat. No. 5,384,261 (in situsynthesis by mechanically directed flow paths); and U.S. Pat. No.5,700,637 (synthesis by spotting, printing or coupling); the disclosureof which are herein incorporated in their entirety by reference. Anothermethod for coupling DNA to beads uses specific ligands attached to theend of the DNA to link to ligand-binding molecules attached to a bead.Possible ligand-binding partner pairs includebiotin-avidin/streptavidin, or various antibody/antigen pairs such asdigoxygenin-antidigoxygenin antibody (Smith et al., Science258:1122-1126 (1992)). Covalent chemical attachment of DNA to thesupport can be accomplished by using standard coupling agents to linkthe 5′-phosphate on the DNA to coated microspheres through aphosphoamidate bond. Methods for immobilization or oligonucleotides tosolid-state substrates are well established. See Pease et al., Proc.Natl. Acad. Sci. USA 91(11):5022-5026 (1994). A preferred method ofattaching oligonucleotides to solid-state substrates is described by Guoet al., Nucleic Acids Res. 22:5456-5465 (1994). Immobilization can beaccomplished either by in situ DNA synthesis (Maskos and Southern, Nuc.Acids Res. 20:1679-84, 1992) or by covalent attachment of chemicallysynthesized oligonucleotides (Guo et al., supra) in combination withrobotic arraying technologies.

In addition to the solid-phase technology represented by biochip arrays,gene expression can also be quantified using liquid-phase arrays. Onesuch system is kinetic polymerase chain reaction (PCR). Kinetic PCRallows for the simultaneous amplification and quantification of specificnucleic acid sequences. The specificity is derived from syntheticoligonucleotide primers designed to preferentially adhere tosingle-stranded nucleic acid sequences bracketing the target site. Thispair of oligonucleotide primers form specific, non-covalently boundcomplexes on each strand of the target sequence. These complexesfacilitate in vitro transcription of double-Stranded DNA in oppositeorientations. Temperature cycling of the reaction mixture creates acontinuous cycle of primer binding, transcription, and re-melting of thenucleic acid to individual strands. The result is an exponentialincrease of the target dsDNA product. This product can be quantified inreal time either through the use of an intercalating dye or a sequencespecific probe. SYBR(r) Green 1, is an example of an intercalating dye,that preferentially binds to dsDNA resulting in a concomitant increasein the fluorescent signal. Sequence specific probes, such as used withTaqMan technology, consist of a fluorochrome and a quenching moleculecovalently bound to opposite ends of an oligonucleotide. The probe isdesigned to selectively bind the target DNA sequence between the twoprimers. When the DNA strands are synthesized during the PCR reaction,the fluorochrome is cleaved from the probe by the exonuclease activityof the polymerase resulting in signal dequenching. The probe signallingmethod can be more specific than the intercalating dye method, but ineach case, signal strength is proportional to the dsDNA productproduced. Each type of quantification method can be used in multi-wellliquid phase arrays with each well representing primers and/or probesspecific to nucleic acid sequences of interest. When used with messengerRNA preparations of tissues or cell lines, an array of probe/primerreactions can simultaneously quantify the expression of multiple geneproducts of interest. See Germer et al., Genome Res. 10; 258-266 (2000);Held et al., Genome Res. 6; 986-994 (1996).

(iv) Measurement of Altered Protein 14-3-3ζ Levels in Cell Extracts, forExample by Immunoassay.

Testing for proteinaceous neoplastic marker expression product in abiological sample can be performed by any one of a number of suitablemethods which arc well known to those skilled in the art. Examples ofsuitable methods include, but are not limited to, antibody screening oftissue sections, biopsy specimens or bodily fluid samples. To the extentthat antibody based methods of diagnosis are used, the presence of theprotein may he determined in a number of ways such as by Westernblotting, ELISA or flow cytometry procedures. These, of course, includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labelled antibodyto a target.

Sandwich assays area useful and commonly used assay. A number ofvariations of the sandwich assay technique exist, and all are intendedto be encompassed by the present invention. Briefly, in a typicalforward assay, an unlabelled antibody is immobilized on a solidsubstrate and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-antigen complex, asecond antibody specific to the antigen, labelled with a reportermolecule capable of producing a detectable signal is then added andincubated, allowing time sufficient for the formation of another complexof antibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample. Variations on theforward assay include a simultaneous assay, in which both sample andlabelled antibody are added simultaneously to the bound antibody. Thesetechniques are well known to those skilled in the art, including anyminor variations as will be readily apparent.

In the typical forward sandwich assay, a first antibody havingspecificity for the marker or antigenic parts thereof, is eithercovalently or passively bound to a solid surface. The solid surface istypically glass or a polymer, the most commonly used polymers beingcellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene. The solid supports may be in the form of tubes, beads,discs of microplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking, covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes) and under suitable conditions (e.g. 25° C.) to allowbinding of any subunit present in the antibody. Following the incubationperiod, the antibody subunit solid phase is washed and dried andincubated with a second antibody specific for a portion of the antigen.The second antibody is linked to a reporter molecule which is used toindicate the binding of the second antibody to the antigen.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody. Alternatively, a second labelled antibody, specificto the first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase and alkaline phosphatase, amongst others.The substrates to be used with the specific enzymes are generally chosenfor the production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody hapten complex, allowed to hind,and then the excess reagent is washed away. A solution containing theappropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of antigen which was present in the sample.“Reporter molecule” also extends to use of cell agglutination orinhibition of agglutination such as red blood cells on latex beads, andthe like.

Alternately, fluorescent compounds, such as fluorecein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. Alter washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

(v) Determining altered protein expression based on any suitablefunctional test, enzymatic test or immunological test in addition tothose detailed in points (iv) above.

(vi) To the extent that one is screening for protein 14-3-3ζ/DISC1complex formation, a variety of methods could be used includingimmobilising one or other molecule on a solid support and then exposingthe immobilised target to the biological sample and screening forcomplex formation. Such screening techniques are generally describedhereinbefore. Co-immunoprecipitation techniques may also conveniently beutilised. Alternatively, a biocore protein interaction system could beused.

As detailed hereinbefore, it would he appreciated by the skilled personthat designing the assays for detecting complex formation may requirethe inclusion of molecules other than just DISC1 and protein 14-3-3ζsince Nedll and LIS1 are also involved in the formation of thefunctional complex. The design of such an assay would be well within theskill of the person in the art.

In the context of these types of screening methods, it would beappreciated that their advantage is that one need not necessarily knowthe nature of the defect which may exist in either or both of theprotein 14-3-3ζ molecule or the DISC1 molecule. Rather, since one ismerely screening for the occurrence of complex formation orco-immunoprecipitation, or not, the identity of the defect becomesirrelevant.

A related aspect of the present invention provides non-human mammals inwhich protein 14-3-3ζ gene expression has been knocked out. Thedevelopment of such animals facilitates the screening for and analysisof therapeutically and/or prophylactically effective proteinaceous ornon-proteinaceous molecules which may modulate the onset and progressionof a neuropsychiatric disorder, such as the schizophrenic phenotype.This development now provides an extremely valuable means for, intercilia, studying the functional role of protein 14-3-3ζ in relation tothe onset of schizophrenia and rationally designing prophylactic and/ortherapeutic treatment regimes.

Accordingly, another aspect of the present invention is directed to anon-human mammal deficient in functional protein 14-3-3ζ in which a geneencoding protein 14-3-3ζ has been deleted.

The term “phenotype” should be understood as a reference to the totalityof the functional and structural characteristics, or any particularcharacteristic or set of characteristics, of an animal as determined byinteraction of the genotype of the organism with the environment inwhich it exists. In the context of the present invention, the subjectphenotype is the onset of schizophrenia, a predisposition to the onsetof schizophrenia or the onset/predisposition to the onset of one or moresymptoms associated with schizophrenia. This phenotype is hereinreferred to as a “schizophrenia phenotype”.

Preferably, said non-human mammal is a mouse.

It should be understood that the present invention also provides cellsand cell lines comprising the 14-3-3ζ knockout described above. Thesecells/cell lines may be derived from any suitable source, or may begenerated by any suitable means.

The development of the mammals (herein referred to as a “l4-3-3ζknockout”) of the present invention now facilitates a wide variety ofhighly useful applications including, but not limited, to screeningmethods to identify agents which mimic protein 14-3-3ζ functionality14-3-3ζ/DISC1 complex formation or otherwise improve the symptoms ofschizophrenic phenotype of these mammals.

Accordingly, yet another aspect of the present invention is directed toa method of screening for an agent which mimics protein 14-3-3ζfunctionality or 14-3-3ζ/DISC1 complex formation or otherwise improvesthe symptoms of a schizophrenic phenotype, said method comprisingadministering to a 14-3-3ζ knockout non-human animal a putativemodulation agent and screening for altered phenotype.

Reference to an “agent” should be understood as a reference to anyproteinaceous or non-proteinaceous molecule derived from natural,recombinant or synthetic sources including fusion proteins or following,for example, natural product screening and which achieves the object ofthe present invention. Synthetic sources of said agent include forexample chemically synthesised molecules. In other examples, phagedisplay libraries can be screened for peptides while chemical librariescan be screened for existing small molecules.

By way or example, diversity libraries, such as random combinatorialpeptide or nonpeptide libraries can be screened. Many publicly orcommercially available libraries can be used such as chemicallysynthesized libraries, recombinant (e.g., phage display libraries) andin vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal. PNAS USA 91: 11422-26 (1991): Houghten et al. Nature 354:84-88(1991); Lam et al. Nature 354:82-84 (1991); Medynski, Bio/Technology12:709-710 (1994); Gallop et al. J. Medicinal Chemistry 37(9):1233-1251(1994); Ohlmeyer et al. PNAS USA 91:9022-9026 (1993); Erb et al. PNASUSA 91:11422-26 (1994); Houghten et al. Biotechniques 13(3):412-421(1992); Jayawickreme et al. PNAS USA 91:1614-1618 (1994); Salmon et al.PNAS USA 90:11708-11712 (1993); international Patent Publication No. WO93/20242; and Brenner and Lerner, PNAS USA 89:5381-3 (1992).

Examples of phage display libraries are described by Scott and Smith,Science 249:386-390 (1990); Devlin et al. Science 249:404-406 (1990);Christian et al. J. Mol. Biol. 227:711-718 (1992); Lenstra, J. Immun.Methods 152:149-157 (1992); Kay et al. Gene 128:59-65 (1993) andinternational Patent Publication No. WO 94/18318.

In vitro translation-based libraries include but are not limited tothose described in Mattheakis et al, PNAS USA 91:9022-9026 (1994).

Without limiting the present invention in any way a test compound can bea macromolecule, such as biological polymer, including polypeptides,polysaccharides and nucleic acids. Compounds useful as potentialtherapeutic agents can be generated by methods well known to thoseskilled in the art, for example, well known methods for producingpluralities of compounds, including chemical or biological moleculessuch as simple or complex organic molecules, metal-containing compounds,carbohydrates, peptides, proteins, peptidomimetics, glycoproteins,lipoproteins, nucleic acids, antibodies, and the like, are well known inthe art and arc described, for example, in Huse, U.S. Pat. No.5,264,563; Francis et al., Curr. Opin. Chem. Biol., 2:422-428 (1998);Tietze et al., Curr. Biol. 2:363-381 (1998); Sofia, Molecule. Divers.3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); andthe like. Libraries containing large numbers of natural and syntheticcompounds also can be obtained from commercial sources. Combinatoriallibraries of molecules can be prepared using well known combinatorialchemistry methods (Gordon et al., J. Med. Chem. 37:1233-1251 (1994);Gordon et al., J. Med. Chem. 37:1 385-1401 (1994); Gordon et al., Ace.Chem. Res. 29:144-154 (1996); Wilson and Czarnik, eds., CombinatorialChemistry: Synthesis and Application, John Wiley & Sons, New York(1997).

Reference to detecting an “altered expression phenotype” should beunderstood as the detection of any form of change associated withmodulation of 14-3-3ζ functioning. These may be detectable, for example,as intracellular changes, changes observed extracellularly (for example,detecting changes in downstream product levels or activities) or changesin the phenotype/condition of the non-human mammalian subject. Forexample, subsequently to administering the agent to the 14-3-3ζ knockoutmouse of the present invention, one may perform one or more of thebehavioural assays described in Example 1, such as:

(i) locomotor function test;

(ii) object recognition test;

(iii) elevated cross bar test;

(iv) escape water maze test; or

(v) PPI test.

If more detailed molecular tests are required to be performed tovalidate or otherwise further investigate phenotypic changes observed atthe behavioural level, then cells or tissues can be harvested from thesubject knockout animals in order to enable more detailed analyses whichare performed at the cellular or molecular level. Alternatively, one maytest cell lines generated from these animals.

It should be understood that these aspects of the present inventionprovide not only a means of screening for novel agents which modulatethe schizophrenia phenotype of individuals exhibiting impaired 14-3-3′functioning, but also provide a means of assessing the benefit/sideeffects of existing treatment regimes in the context of this group ofpatients.

The present invention is further described by reference to the followingnon-limiting examples.

EXAMPLE 1

Materials and Methods

Mice. 14-3-3ζ^(Gt(OST062)Lex) and 14-3-3ζ^((Gt(OST390)Lex) mutant micecarrying gene trap constructs that contain the Geo reporter gene werederived from Lexicon Genetics ES cell lines OST062 and OST390,respectively. The gene trap vector in 14-3-3ζ^(Gt(OST062)Lex) miceinserted into the first intron of 14-3-3ζ, whereas the gene trap vectorin 14-3-3ζ^(Gt(OST390)Lex) mice inserted into the second intron of14-3-3ζ. ES cell lines were amplified and injected into SV129blastocysts. Resulting germ line transmitting males were eithermaintained in the SV129 background, or backcrossed in to the C57/B16 andBALBC backgrounds over 6 generations, qRT-PCR and western blot fromwhole tissue samples was used to confirm complete KO of the gene inthese mouse strains, 14-3-3C genotype was determined by PCRamplification of genomic tail DNA using the primers detailed insupplementary table 1. The WT allele amplified a band of 288 bp(14-3-3ζ^(Gt(OST062)Lex)) or 445 bp (14-3-3ζ^(Gt(OST390)Lex)) and themutant gene trapped allele amplified a band of 165 bp(14-3-3ζ^(Gt(OST062)Lex)) or 203 by (14-3-3ζ^((gt(OST390)Lex)). Micewere maintained as heterozygous breeding pairs that were phenotypicallyindistinguishable to WT littermates. Animal experiments were conductedin accordance with the guidelines of the Animal Ethics Committee of theinstitute of Medical and Veterinary Sciences and the University ofAdelaide.Behavioural assays. All procedures were carried out under normal lightconditions between 8.00 am and 12.00 pm. Behavioural phenotyping wasperformed as previously described (Coyle et al. Behav Brain Res 2009,197(1): 210-218; Summers et al. Pediatr Res 2006; 59(1): 66-71; van denBuuse et al. Int J Neuropsychopharmacol 2009; 12(10):1383-1393). Onecohort of mice was used for the open field test at ages of 5-, 10-, 20-and 40-week time points. One cohort of mice was used at the age of 12weeks for spatial working memory, then elevated plus maze and objectrecognition tasks. A separate cohort of mice was used at the age of 12weeks for PPI.Locomotor function test. Exploratory activity and anxiety level of micewere measured in an open field made from a box (50 cm×27 cm) with thefloor divided into 15 squares (9 cm×10 cm). Each mouse was introduced into the same position of the box facing the right top corner. Thebehaviour of the Mouse was observed for 3 min and locomotor activity wasscored as a measure of line crossings (i.e. when a mouse removed allfour paws from one square into another). Number of rears up was scoredwhen a mouse had both front paws off the floor. Urine and faecalmaterial were removed between session and the box was cleaned thoroughlywith 80% ethanol to remove any lingering scents.Object recognition test. The object recognition task takes advantage ofthe natural affinity of mice for novelty; mice that recognise apreviously seen (familiar) object will spend more time exploring novelobjects (Dere et al. Neurosci Biobehav Rev 2006; 30(8):1206-1224; Sik etal. Behav Brain Res 2003; 147(1-2):49-54). Briefly, the apparatusconsisted of a plastic arena (length; 50 cm, width; 35 cm, depth; 20 cm)filled with bedding. Two different sets of objects were used; ayellow-capped plastic jar (height, 6 cm; base diameter, 4.3 cm) and ared plastic bulb (length: 8 cm, width: 4 cm). Mice spent equal amountsof time when presented with both of these objects, regardless of theposition they-were placed in the arena (data not shown). At 12 weeks ofage the same cohort of mice tested for spatial learning and memory wereassessed for object recognition memory. Each mouse was given 5-min toexplore the test box without any objects present to habituate them tothe test arena. Mice underwent the testing session comprised of twotrials. The duration of each trial was 3 min. During the first trial(the sample phase), the box contained two identical objects (a, samples)which were placed in the north-west (left) and northeast (right) cornersof the box (5 cm away from the walls). A mouse was always placed in theapparatus facing the south wall. After the first explorationperiod,-mice were placed back in their homecage. After a 15-minretention interval, the mouse was placed in the apparatus for the secondtrial (choice phase), but now with a familiar one (a. sample) and anovel object (b). The objects were cleaned thoroughly with alcoholbetween sessions to remove any lingering scents. The time spentexploring each object during trial 1 and trial2 was recorded.Exploration was defined as either touching the object with the nose orbeing within 2 cm of it. The basic measures in the object recognitiontask were the times spent exploring an object during trial land trial 2.Several variables were measured during the tests: e1 (a+a) and e2 (a+b)are measures of the total exploration time of both objects during trial1 and trial 2, respectively. h1 is an index of habituation measured bythe difference in total exploration time from trial 1 to trial 2(e1-e2).d1 (b−a) and d2 (d1/e2) were considered as index measures ofdiscrimination between the novel and the familiar objects. Thus, d2 is arelative measure of discrimination that corrects d1 for exploratoryactivity (c2). A discrimination index above zero describes animalsexploring the novel object more than the familiar object. An animal withno preference for either object will have an index near zero. Mice witha total exploration time of less than 7 s during trials in the sample orchoice phase were excluded from the analyses as the measurement ofexploration time has been found to be non-reliable below this threshold(van den BUUSC et al. supra; de Bruin et al. Pharmacol Biochem Behav2006; 85(1):253-260).Elevated cross bar test. The anxiety behaviour of mice based on theirnatural aversion of open and elevated areas was assessed using anelevated plus-maze as previously described (Komada et al. J Vis Exp2008; (22); Walf et al. Nat Protoc 2007; 2(2):322-328). Briefly, theapparatus was made in the shape of a cross from black plexiglass andconsisted of two open ants (25 cm×5 cm) and two closed arms (25 cm×5cm×16 cm) that crossed in the middle perpendicular to each other. In themiddle of the to arms there was a central platform (5 cm×5 cm). Thecross maze was raised 1 m from the ground. Individual mice wereintroduced to the center of the apparatus facing the open arm oppositeto the experimenter were and observed by video recording for 5 minutes.The number of entries into the open and closed arms and the time inexploring both types of arm were scored. Naturalistic behaviour of themouse such as the number of head dipping, number of rearing and numberof stretch attended postures were measured. After each trial all armsand the central area thoroughly cleaned with alcohol to remove anylingering scents.Escape water maze test. Spatial learning and memory was assessed using across-maze escape task as previously described (Coyle et al. 2009,supra). The cross maze was made of a clear plastic (length, 72 cm; armdimensions, length 26cm×width 20 cm) and placed in circular pool ofwater (1 m diameter) maintained at 23 C. Milk powder was mixed into thewater to conceal a submerged (0.5cm below the water surface) escapeplatform placed in the distal north arm of the maze. The pool wasenclosed by a black plastic wail (height, 90 cm). Constant spatial cueswere arranged at each arm of the maze and by the experimenter who alwaysstood at the southern end during the training and testing procedures 12week old mice were individually habituated to the maze environment bybeing placed into the pool without the escape platform and allowed toswim for 60 s. Learning trials were conducted over a 6-day trainingperiod in which mice were required to learn the position of thesubmerged escape platform from the other three (East, South, West) armsthat did not contain an escape platform. Each mouse was given six dailytrials (two blocks of three trials separated by a 30 min rest interval),in which each of the three arms were chosen as a starting point in arandomized pattern (twice daily). For each trial, the mouse was placedin the distal end of an arm facing the wall and allowed 60 s to reachthe escape platform where it remained for 10 s. Mice that did not climbonto the escape platform in the given time were placed on the platformfor 10 s. The mouse was then placed in a cage for 10 s and subsequenttrials were continued. Mice were assessed on their long-term retentionof the escape platform location which was placed in the same position asduring the learning phase. Memory was tested 14 (M1) and 28 (M2) daysafter the final day of learning and consisted of a single day of 6trials as described for the learning period. Data were recorded for eachmouse for each trial on their escape latency (i.e. time (s) taken toswim to the platform), number of correct trials (i.e. if a mouse foundthe platform on the first arm entry) and number of incorrectentries/reentries (i.e. the number of times that a mouse went into anarm that did not contain the escape platform).PPI test. Startle, startle habituation and PPI of startle were assessedusing, an eight-unit automated system (SR-LAB, San Diego Instruments,USA) as previously described (van den Buuse et al. 2009 supra). Briefly,mice were placed in clear Plexiglas cylinders which were closed oneither side and acoustic stimuli were delivered over 70-dB backgroundnoise through a speaker in the ceiling of the box. Each testing sessionconsisted of 104 trials with an average inter-trial interval between 25s. The first and last eight trials consisted of single 40-ms 115 -30 dBpulse alone startle stimuli. The middle 88 trials consisted ofpseudo-randomised delivery of 16 115-dB pulse-alone stimuli, eighttrials during which no stimulus was delivered, and 64 prepulse trials.The total of 32 115-dB pulse alone trials was expressed as four blocksof eight and used to determine startle habituation. Prepulse trialsconsisted of a single 115-dB pulse preceded by a 30-ms or 100-msinter-stimulus interval (ISI) with a 20-ms non-startling stimulus of 2,4, 8 or 16 dB over the 70-dB baseline. Whole-body startle responses wereconverted into quantitative values by a piezo -electric accelerometerunit attached beneath the platform. Percentage prepulse inhibition (%PPI) was calculated as pulse-alone startle response—prepulse+pulsestartle response/pulse-alone startle response×100.Statistical analysis. All statistical calculations are presented asmean±SEM and were performed using SAS Version 9.2 (SAS Institute Inc.,Cary, N.C., USA). For open field data the number of line crossings werecompared across the WT and mutant groups and over time using a linearmixed effects model. A random mouse effect was included in the model toaccount for the dependence in repeated observations from the same mouse.Data from the elevated cross bar, was compared between WT and mutantsusing an independent samples t-test. For the water cross-maze testescape latency was compared between the two treatment groups and overtime using a Cox proportional hazards model. Robust variance estimationwas used in the model to adjust for the dependence in results due torepeated measurements on the same mouse. In the model group (WT or KO),time (days 1 to 6) and the interaction between group and time wereentered as predictor variables. Escape latency was considered rightcensored at 30 seconds when a mouse had yet to find the exit. In ourstudy there were too many animals with art escape latency censored at 30seconds to be able to treat the outcome as being normally distributed.Thus it was not feasible to use a linear mixed effects model. Incorrectentries were compared between WT and mutant groups and over time using anegative binomial regression model. In the model group (WT or KO), time(days 1 to 6) and the interaction between group and time were entered aspredictor variables. A generalised estimating equation was used toaccount for the dependence in results one to repeated measurements onthe same mouse. Data from the PPI tests were compared using two-wayanalysis of variance (ANOVA) with repeated measures (Systat, version9.0, SPSS software: SPSS Inc., USA). For this analysis the between-groupfactor was genotype and the within group, repeated-measures factors wereprepulse intensity and startle block. In all studies ap value of <0.05was considered to he statistically significant.Immunohistochemistry. Sections were blocked in 10% non-immune horseserum in PBST (0.1 M PBS, (1.3% Triton X-100, 1% BSA) for I hat roomtemperature (RT) and subsequently incubated with primary antibodiesovernight at RT. Primary antibodies and dilutions: rabbit polyclonal to14-3-3ζ (1:200) (Guthridge et al. Blood 2004: 103(3):820-827), rabbitpolyclonal to 0-tubulin (1:250, Sigma), rabbit polyclonal tocalbindin-D28K (1:1000, Chemicon), mouse monoclonal to NeuN (1:500,Chemicon), rabbit polyclonal to synaptophysin (1:100, Cell Signaling).On the following day, sections were incubated with secondary antibodiesfor 1 h at RT. After 3 times 0.1M PBS wash, the sections were mounted inProlong® Gold antifade reagent with DAPI (Molecular Probes).BrdU-pulse-chase analysis and TUNEL labelling, BrdU was injected at 100μg/g of body weight of the pregnant mice at 14.5 dpc or 16.5 dpc and thepups were euthanized at postnatal-day-7. Final destination of theproliferating hippocampal neurons that were born at these time pointswere revealed by BrdU immunohistochemistry on frozen brain sections.Tissue were denatured with 2M HCl for 20 min at 37° C., neutralised in0.1 M borate buffer (pH 8.5) for 10 min, blocked with 10% horse serum inPBST and probed with rat monoclonal anti-BrdU (1:250; Abcam) and mousemonoclonal anti-NeuN (1:500; Chemicon) antibodies overnight at 4° C.Cell apoptosis was determined by the TUNEL assay using the In Situ CellDeath Detection Kit (TMR Red; Roche Applied Science) according to themanufacturer's instructions followed by counterstained with DAPI(Molecular Probes).Immunoprecipitation. All protein extracts were prepared by lysis inNP40lysis buffer composed of 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10%glycerol, 1% Nonidet P-40, and protease and phosphatase inhibitors (10mg of aprotinin per m1,-10 mg of leupeptin per ml. 2 mMphenylmethylsulfonyl fluoride, and 2 mM sodium vanadate). Samples werelysed for 60 min at 4 C, then centrifuged at 10,000 g for 15 min. Thesupernatants were precleared with mouse Ig-coupled Sepharose beads for30 min at 4 C. The precleared lysates were incubated for 2 h at 4 C with2 ug/ml of either anti-DISC1 antibody (C-term) (In vitrogen) oranti-14-3-3 antibody (3F7 Abeam) absorbed to protein A-Sepharose(Amersham Biosciences). The sepharose beads were washed 3 times withlysis buffer before being boiled for 5 min in SDS-PAGE sample buffer.The immunoprecipitated proteins and lysates were separated by SDS-PAGE,and clectrophorectically transferred to a nitrocellulose membrane andanalysed by immunoblotting.Immunoblotting. The membranes were probed with either anti-14-3-3ζ EB1pAb at 1:1000 (Guthridge et al. 2004 supra) or anti DISC1 (C-term)(Invitrogen) at 1 ug/ml). For analysis of 14-3-3ζ from brain tissuerabbit polyclonal against the (3-actin (1:5000, Millipore) was used as aloading control. Bound antibodies were detected with HRP-conjugatedsecondary antibody (1:20,000, Pierce-Thermo Scientific). Immunoreactiveproteins were visualized by ECL (Luminescent image Analyzer LAS-4000.Fujifilm, Japan). The images were analysed with Multi Gauge Ver3.0(Fujifilm, Japan).Neuronal cell cultures. P7 hippocampi neuron-glial cocultures wereprepared as described (Kaech et at, Nat Protoc 2006, 1(5):2406, 2415).Nitric acid-treated coverslips (diameter 13 mm) were coated with 100ug/ml poly-L-lysin/PLL (Sigma) in borate buffer for overnight at 37° C.,and were then washed with sterile water for 3×1 h. Dentate gyri and CAsamples were dissected and dissociated in Hank's balanced salt solution(HBSS) and neurons were plated at a density of 1×10⁵ cells per culturedish (with 4 PLL-coated coverslips). Cultures were incubated for 7 and14 days in vitro for neurite outgrowth assay. Cells were fixed in 4% PFAfor 1 h, preincubated in 10% non-immune horse serum in PBST (0.1M PBS,0.1% Triton X-100, 1% BSA) for 1 h at room temperature (RT) andincubated overnight at 4° C. with primary antibodies against mousemonoclonal MAP2 (1:200. Millipore) and 14-3-3ζ, (1:1000). The coverslipswere then incubated with the corresponding secondary antibodies for 1 hat RT. Coverslips were mounted with anti -fade DAPI (Molecular Probes).Results14-3-3ζ, Mutant Mice Display Behavioural and Cognitive Defects

14-3-3 proteins are abundantly expressed in the developing and adultbrain (Berg et al, Nat Rev Neurosci 2003; 4(9):752-762; Baxter et al.Neuroscience 2002; 109(1):5-14). To ascertain the role of 14-3-3ζ inneurodevelopment and brain function generated two knockout mouse lineswere generated from embryonic stem cell clones containing retroviralgene-trap insertions within intron 1 or 2, termed 14-3-3 and14-3-3ζ^(Gt(OST390)Lex), respectively (FIG. 8; Lexicon Genetics):Quantitative RT-PCR and western blot on embryonic and adult brain tissuefrom heterozygous intercrosses confirmed that the gene trap vectorsdisrupted gene transcription and created null alleles (FIG. 9). Thesemutant lines are referred to as 14-3-3ζ^(062+/−) and 14-3-3ζ^(390+/−).Unlike deletions of other 14-3-3 isoforms (Su et al. Proc Natl Acad SciUSA 2011; 108(4):1555-1560), expression analysis further determined thatremoval of 14-3-3C is not compensated by increased expression of other14-3-3 family members in mutant mice (FIG. 10). Inter crosses of14-3-3(heterozygous mice from both strains gave rise to homozygousmutants in the predicted Mendelian ratio (WI 23%, Het 56%, Mut 21%;n-494, p<0.001) indicating that removal of the gene is not embryoniclethal. initial inspection of mutant embryos and newborn mice suggestedthat development proceeded normally as they were morphologicallyindistinguishable from their littermates. However, by P14 mutant micefrom both lines showed growth retardation and by P21 around 20% ofmutant mice had died (WT 29%, Het 54%, Mut 17%; n-1619). The remainingmutant mice were smaller than WT ittermates but had similar lifeexpectancy (P100; WT 24.55+1.7 g. Mut 19.73 g±2.5g). Mutant miceappeared outwardly normal and healthy with no differences in theolfactory test, visual test and wire-hang test.

To definitively analyse the association of 14-3-3ζ with neurologicaldisorders and brain functions, a series of behavioural tests on mutantand control mice were completed. The response of 14-3-3ζ^(062−/−) miceto an open field environment was first evaluated. Mutants showed asignificant increase in distance travelled over the test period that wasmaintained throughout all testing ages (5, 10, 20 and 30 weeks),indicating that mutant mice arc. hyperactive (FIG. 1.A). This effect wassimilar for both males and females with no sex bias (p>0.05).

The mouse's natural exploratory preference of novel objects rather thanfamiliar objects was exploited to test recognition memory. Correctfunctioning of the perirhinal cortex in the medial lobe is essential forthis task (Dere et al. 2006 supra; Sik et al. 2003 supra; Forwood et al.Hippocampus 2005; 15(3):347-355; Winters et al. J Neurosci 2005;25(17):4243-4251). In the sample phase, mice spent an equal timeexploring each identical object (14-3-3ζ^(062+/+), 50.82±1.2%;14-3-3ζ^(062−/−)49.18+1.2%). When presented with a familiar and newobject, 14-3-3ζ^(062−/−) mice exhibited significantly impaired novelobject recognition compared to controls over the test period. Consistentwith a lack of preference between the familiar and novel objects,14-3-3^(062−/−) mice had a reduced discrimination index (time exploringnovel object−time exploring familiar object/time exploring novelobject+time exploring familiar object) indicating that they failed toretain new information (14-3-3ζ^(062+/+), 0.1667±0.086 s;14-3-3^(0624−/−), −0.0569±0.047 s; p<0.05). Once again, there were nosex differences in either phase of testing (p>0.5). Notably,.14-3-3ζ^(062−/−) mutants also demonstrated hyperactivity in the objectrecognition test with longer exploratory times in both phases of thetrial (Sample phase, 14-3-3ζ^(062+/+), 27.33±2.7 s; 14-3-3ζ^(062−/−),38.62±4.1 s; p<0.05: test phase, 14-3-3ζ^(062+/+), 24.58+3.1 s;14-3-3ζ^(062−/−), 50.77±4.7 s; p<0.0001).

The elevated plus maze is widely used to test anxiety behaviour ofrodents (Komada et al; 2008 supra; Walf et al. 2007 supra; Lister R G,Psychopharmacology (Berl) 1987; 92(2):180-185). When placed in such atest, 14-3-3ζ^(062−/−) mice also demonstrated increased activitycompared to wild type controls. 14-3-3ζ^(062−/−) mice had 25.23±1.76transitions between cross arms during a 5 min test period while14-3-3ζ^(062+/+) had 12.29±1.21 (p<0.0001). In addition,14-3-3ζ^(062−/−) mice spent significantly more time in the open arms(FIG. 1B: 114.8±11.5 s) compared to 14-3-3ζ^(062+/+) mice (31.4±6.0 s,p<0.0001), entered them more often (14-3-3ζ^(062+/+), 4.6+0.6; 14-3-3ζ^(062−/−), 15.5±1.7, p<0.0001) and head dipped more,(14-3-3ζ^(062+/+)19.6±1.5; 14-3-3ζ^(062−/−), 33.4±2.4 p=0.0041)suggesting that they had lower levels of anxiety.

Spatial working memory-dependent learning was examined using a crossmaze escape task (Summers et al. 2006 supra). Appropriate signallingbetween the hippocampus and prefrontal cortex are a prerequisite foracquisition of this task. Mice were trained over 6 days to identify thecorrect arm of a cross maze containing a submerged escape platform. Eacharm of the cross maze was denoted by a novel visual cue throughout theexperiment. Although sonic 14-3-3ζ^(062−/−) mice learnt to identify thecorrect arm, they showed increased latency in reaching the platform overthe course of the acquisition period (FIG. 11; χ²(5)=29.8808; p<0.0001)and had significantly decreased arm choice accuracy (FIG. 1C: IRR 0.52;p<0.0001). Their ability to remember the correct cross-arm was thentested by resting them for 14 days or 28 days post acquisition followedby re-testing in the escape platform water maze (M1 and M2,respectively). In comparison to the learning phase, 14-3-3ζ^(062+/+)mice showed no change in escape latency (HR=1.18, p=0.383), whilst14-3-3ζ^(062−/−) demonstrated significantly increased escape latency(HR=2.98, p<0.0001). Consistent with dysfunction inhippocampus-dependent memory, mutant mice also had a significantdecrease in arm choice accuracy (FIG. 1C: IRR=0.231; p<0.0001). Allcognitive defects were independent of sex.

Defects in sensorimotor gating are an endophertotype of neuropsychiatricdisorders such as schizophrenia and related disorders. Appropriatesignalling in the hippocampus and other brain regions are essential forthis filtering mechanism. To determine if 14-3-3ζ, mutant mice haveabnormal sensorimotor gating, prepulse inhibition (PPI) of the acousticstartle reflex was assessed. It was found that 14-3-3^(062−/−) mice hada significantly lower PPI (FIG. 1D: main effect of genotypeF(1,20)=5.89, p=0.025) and startle (FIG. 12: F(1,20)=5.87, p=0.023)compared to 14-3-3ζ^(062+/+) mice. Increasing levels of prepulseintensities caused, similar increases in PPI in WT and mutant mice (FIG.1D). Overall, startle amplitudes were reduced in mutant mice but startlehabituation was normal (FIG. 12).

14-3-3ζ, is Expressed in Hippocampal Neurons to Control Lamination

To determine if the cognitive and behavioural deficits arise fromneurodevelopmental defects of the hippocampus, the role of 14-3-3ζ inneuronal development was analysed. Hippocampal neurons derive from theneuroepithelium along the ventricular zone (NEv) and from a restrictedarea of neuroepithelium adjacent to the fimbria (NEf) (Nakahira et al. JComp Neurol 2005; 483(3):329-340) (FIG. 2A). At 14.5 dpc 14-3-3ζimmunostaining was detected in migrating hippocampal neurons within theintermediate zone, but not in their neuroepithelial precursors (FIG.2Bi). By P0 14-3-3ζ immunostaining was also detected in pyramidal cellsof the hippocampal proper/cornu ammonis (CA) (FIG. 2Biii), Takingadvantage of the Beta-geo,transgene within the gene trap vectors of the14-3-3ζ mouse lines endogenous expression of 14-3-3ζ with B-galactoidasestaining in heterozygous mice was monitored. Consistent withimmunostaining, expression of 14-3-3ζ at the transcript level inmigrating CA neurons was identified. In addition, expression within CAand DG neurons was detected into late adulthood (FIG. 2C). Unexpectedly,however, 14-3-3ζ was undetectable in other regions of brain, such as thecerebellum, after early post natal stages (FIG. 13). Expression withinCA and DG neurons was confirmed by western blot of protein extractedfrom microdissected adult hippocampi (FIG. 2D). This also confirmedcomplete removal of the protein from these brain regions of14-3-3ζ062−/− mice. Finally, after 10 days in vitro (DIV), hippocampalMAP2 positive neuronal cultures also showed punctate immunocytostainingfor 14-3-3ζ within the cell body and axon/dendrites (FIG. 2E).

As 14-3-3ζ is expressed in hippocampal neurons we next examined if CAand DG neurons were examined to determine if they are positionedcorrectly in adult and embryonic mutants. Nissi-staining of14-3-3ζ^(062−/−) mice revealed developmental defects first noticeableprior to hippocampal maturation (5/5 at P0, 4/4 at P7, 2/2 at P28 and2/2 at P56; FIG. 3A and FIG. 14). Specifically, pyramidal neurons wereectopically positioned in the stratum radiatum and stratum oriens inaddition to their usual resting place of the stratum pyramidale. Withinthe CA3 subfield, pyramidal neurons split in to a bilaminar stratuminstead of a single cell layer. Dentate granule neurons were alsodiffusely packed in the 14-3-3ζ^(062−/−) mice compared with14-3-3ζ^(062+/+) littermates. Consistent with Nissl staining, analysisof hippocampal organization in thy1-YFP mice also revealed a disruptedlaminar organization (FIG. 3B).

Consideration was then directed to whether ectopically positionedpyramidal cells developed into mature neurons. In all 14-3-3ζ^(062−/−)hippocampi (4/4 pups) ectopic cells were positive for the neuronalmarker NeuN (FIG. 3C). Rather than positioning themselves in the deepmolecular layer, neurons also matured in the superficial layer of CA3.Together, this data infers that mispositioned cells in the hippocampusform functional pyramidal and granular neurons: Additionally, TUNELstaining of hippocampi from embryonic, early postnatal and adult miceshowed no apparent differences between genotypes (FIG. 15) suggestingthat lack of 14-3-3ζ does not affect neuronal viability.

14-3-3ζ-deficient Mice Display Hippocampal Neuronal Migration Defects

The expression of 14-3-3ζ within the intermediate zone at 14.5 dpc andthe presence of mature neurons in the superficial layer at P0 raised thepossibility that the aberrant laminar structure may arise frotherroneous migration. To visualize hippocampal neuron migration, BrdUbirthdating was completed by injecting BrdU into pregnant dams fromheterozygous 14-3-3ζ⁰⁶² crosses at 14.5 dpc and 16.5 dpc.14-3-3ζ^(062+/+) and 14-3-3ζ^(062−/−) pups were collected at P7 andBrdU-retaining cells were identified in coronal sections. Sections werecounterstained with DAPI to identify separate layers of the hippocampus.BrdU-retaining cells were counted from 10 μm sections using 5 mice ofeach genotype and the relative percentage in each layer was quantified.Both injection time points show that nearly all neurons born in theventricular zone at 14.5 dpc or 16.5 dpc migrate in to the stratumpyramidale of the CA in control mice (FIG. 4). Strikingly, however, asignificant percentage of BrdU-retaining cells were identified outsideof the stratum pyramidale in 14-3-3ζ^(062−/−) mice. Failure of neuronsto migrate from their birthplace or to stop within their correct layertherefore gives rise to the duplicated stratum pyramidale in the14-3-3ζ^(062−/−) hippocampus.

Functional Disrupted Mossyfibre Circuit and Aberrant Synaptic Terminalsin Pyramidal Cells in 14-3-3ζ-Deficient Mice

Communication between the CA3 pyramidal neurons and DG granule cells isachieved through precise axonal navigation and synaptic targeting. Theissue of whether misaligned pyramidal neurons affected the hippocampalcircuit was assessed by performing immunohistochernical staining withanti-calbindin in P0, P7 and P56 hippocampi. In control mice, mossyfibres sprouted from the somata of the granule cells and bifurcated intoinfrapyramidal mossy fibre (IPMF) and suprapyramidal mossy fibre (SPMF)tracts spanning the stratum pyramidale of CA3 (FIG. 5). In14-3-3ζ^(062−/−) mice the IPMF tract navigated along the apical surfaceof CA3 pyramidal neurons, however, the SPMF tract was misrouted amongstthe CA3 neurons.

To determine whether DG granular cells synapsed on their CA targetcells, anti-synaptophysin was used to identify presynapses in both the1PMF and SPMF of the CA3 subfield in control animals. In14-3-3ζ^(062−/−) mice, misrouted axons also formed aberrant synapseswithin the stratum pyramidale (FIG. 6). Visualisation of synapticboutons by golgi stain further revealed notable differences in synapseformation in CA3. In control animals large spine excrescences on theproximal region of the apical dendrites were followed by fine-calibredendritic branches. In pyramidal neurons of 4-3-3ζ^(062−/−) mice thedendritic tree appeared to have similar numbers of branch points but hadthorny excrescences from the misrouted mossy fibre tracts on bothproximal and distal apical dendrites of all mice examined.

To identify the molecular pathways employed by 14-3-3ζ to coordinateneuronal migration and axonal pathfinding co-immunoprecipitationexperiments were performed on whole brain extracts from P7 mice. It wasfound that 14-3-3ζ could be co-immunoprecipitated with an antibodyraised to the C-terminus of DISC1. Vice versa, it was also found thatDISC1 could be co-immunoprecipitated with an antibody recognising14-3-3ζ (FIG. 7). Surprisingly, the data indicate that 14-3-3ζ interactsspecifically with the 75 kDa form of DISC1 rather than the 100 kDa fulllength protein, indicating that DISC1 functions in an isoform specificmanner in neurodevelopment.

EXAMPLE 2

Converging clinical and experimental evidence suggest that schizophreniaand related disorders arise from interconnected defects in thedopaminergic and gutamatergic neurotransmitter pathways. The hippocampushas been posited as a key structure in this model as hippocampalpyramidal neurons integrate the glutamatergic and dopaminergic systems.To determine if either neurotransmitter pathway underpins theschizophrenia-like behavioural defects in 14-3-3ζ^(−/−) mice,psychotropic drug induced behavioural studies were undertaken thatspecifically antagonize each pathway (FIG. 6). It was found that theNMDA receptor antagonist, MK801, disrupted PPI in wild-type controls butnot in 14-3-3ζ^(−/−) mice. In contrast, the dopamine releaser,apomorphine, had a similar effect on FPI in both 14-3-3ζ^(−/−) mice andwild type controls. This indicates that the baseline PPI defect of14-3-3ζ^(−/−) mice results from deficiencies in the glutamatergicpathway. The hyperdopaminergic hypothesis was also investigated usinganother dopamine releaser, amphetamine, in the locomoter function test.It was found that an enhanced effect occurred in 14-3-3ζ^(−/−) micecompared to wild-type controls (i.e. a decrease in time to becomehyperactive and an increase in distance travelled) indicating that thebaseline hyperactivity of 14-3-3ζ^(−/−) mice results from deficiency inthe dopaminergic pathway (FIG. 17). Thus, 14-3-3ζ^(−/−) mice havedefects in the dopaminergic and glutamatergic neurotramitter pathways.Given the ability of these drugs to induce similar effects inschizophrenia patients, these findings provide solid support for14-3-3ζ^(−/−) mice as a model for schizophrenia and related disorders.

EXAMPLE 3

Reduced dendritic branching and spiny synapses are an anatomicalhallmark of schizophrenia and related disorders. Using complimentarytechniques such as golgi impregnation, in vitro culture of pyramidalneurons and biolistic labelling, analysis has been performed ofdendritic spine numbers and spine size in granular and pyramidal neuronsof the hippocampus (FIG. 18). In strong support of the 14-3-3ζ−/− miceas a robust model for schizophrenia and related disorders, significantlyreduced spines in the hippocampus were found.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 1 Primers used for quantitative RT-PCR, of 14-3-3 isoforms and forgenotyping mice Forward Reverse 14-3-3⁰⁶² WT GAACTTCAGATCTGGTGACGATTGTACTCAAAATGGTGGAC (SEQ ID NO: 2) (SEQ ID NO: 3) 14-3-3ζ⁰⁶² KOGCGTTACTTAAGCTAGCTTGC GATTGTACTCAAAATGGTGGAC (SEQ ID NO: 4) (SEQ ID NO:5) 14-3-3~⁹⁰ WT ACGGCGGGGGGCAGCCAG CTCCTGGAAAGATGCGAAC (SEQ ID NO: 6)(SEQ ID NO: 7) 14-3-3ζ⁹⁰ KO GCGTTACTTAAGCTAGCTTGC CGCTGGGGACCCCGTGC (SEQID NO: 8) (SEQ ID NO: 9) Beta GCAACGATGTGCTGGAGC GAGTTGGACACCGTGGTTTG(SEQ ID NO: 10) (SEQ ID NO: 11) Epsilon TACGACGAAATGGTGGAATCGCTCAGTTTCAACCATTTGC (SEQ ID NO: 12) (SEQ ID NO: 13) EtaCGAGTCGCGAGCGACATG CCCTCCAAGAAGATCGCCTG (SEQ ID NO: 14) (SEQ ID NO: 15)Gamma GCCATGAAGAACGTGACC TCTCGTACTGGGTCTCGC (SEQ ID NO: 16) (SEQ ID NO:17) Sigma TGTGTGCGACACTGTGCTC TCGGCTAGGTAGCGGTAGT (SEQ ID NO: 18) (SEQID NO: 19) Tau TCGCCATGGAGAAGACCG CCGATAGTCCTTGATCAGC (SEQ ID NO: 20)(SEQ ID NO: 21) Zeta GCAACGATGTACTGTCTC CTGGTCCACAATTCCTTTC (SEQ ID NO:22) (SEQ ID NO: 23)

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The invention claimed is:
 1. A method of detecting the level of 14-3-3ζRNA in a biological sample obtained outside the brain comprising:obtaining a biological sample from outside of the brain from a subject;and analyzing the biological sample for expression of 14-3-3ζ RNA usingquantitative reverse transcriptase polymerase chain reaction ormicroarray analysis, wherein said subject has or is suspected of havingschizophrenia, schizotypal personality disorder, psychosis, bipolardisorder, manic depression, affective disorder, schizophreniform,schizoaffective disorders, psychotic depression, autism, drug inducedpsychosis, delirium, alcohol withdrawal syndrome or dementia inducedpsychosis.
 2. The method according to claim 1, wherein the analyzingstep comprises a quantitative reverse transcriptase polymerase chainreaction.
 3. The method according to claim 1, wherein said biologicalsample is a blood sample.
 4. The method according to claim 1, whereinsaid subject is a human.
 5. The method according to claim 1, wherein thebiological sample is cerebrospinal fluid, peripheral blood, or adultderived neural stem cells from dental pulp, hair follicle, or nasal pit.